Biomedical subjects

M Geffard

Publications and source records attributed to M Geffard.

At least 109 records · Page 6Linked to original sources

PubMed1989

Endogenous L-dopa in the rat dorsal vagal complex: an immunocytochemical study by light and electron microscopy.

The aim of this work was to examine L-DOPA immunoreactivity (L-DOPA-IR) in the dorsal vagal complex (DVC) of the rat medulla oblongata containing A2/C2 catecholaminergic cell groups, in order to further evaluate the previously proposed hypothesis that various pools of endogenous L-DOPA could be immunocytochemically demonstrated in the mammalian brain. For this purpose, L-DOPA-IR was studied in DVC in comparison with both some other catecholaminergic areas and dopamine immunoreactivity (DA-IR) on adjacent sections of the same brain, by using specific antibodies against glutaraldehyde conjugated L-DOPA and DA. Also, the first preliminary observations of L-DOPA-IR in DVC neurons at the ultrastructural level are reported. The following main results were obtained: (1) bright, intense and homogeneous L-DOPA staining was found in perikarya and proximal neuronal processes situated within the rostrocaudal extension of the DVC; (2) this staining pattern was readily distinct from weak and heterogeneous DA staining; (3) an inverse L-DOPA/DA staining pattern ratio was identified between the DVC and the mesencephalon; (4) L-DOPA-IR at electron microscopic level was roughly similar to that previously observed for DA-IR in mesencephalic cells and their presumptive projections. Although some discrepancies were noticed between L-DOPA staining and data from the literature on tyrosine hydroxylase labeling, our results could not invalidate the hypothesis that, among high L-DOPA/DA ratio containing neurons, some cells in the DVC may contain only L-DOPA.

Animals↗

PubMed1989

Dopamine and motor activity in the lobster Homarus gammarus.

Motor activity similar to agonistic behaviour is obtained after dopamine (DA) injection in lobster. Specially vigorous swimmeret beatings are observed and can be compared to the 'in vitro' motor activity elicited by DA superfusion of the isolated abdominal nervous system. DA-immunoreactive neurons stained by monoclonal antibodies in abdominal ganglia may be involved in swimmeret activation during the agonistic behavior.

Animals↗

PubMed1989

Demonstration of GABAergic cell bodies in the suprachiasmatic nucleus: in situ hybridization of glutamic acid decarboxylase (GAD) mRNA and immunocytochemistry of GAD and GABA.

The existence of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) was demonstrated by three specific markers; mRNA coding for glutamic acid decarboxylase (GAD) and visualized by in situ hybridization using a 35S-labelled cDNA probe, and GAD protein and GABA were identified by immunocytochemistry using specific antisera. In situ hybridization demonstrated well labelled GAD mRNA positive cells throughout SCN, and GABA and GAD immunoreactive cells showed similar distributions. These results indicate that GABA is a transmitter of a large portion of the SCN neuronal population.

Animals↗

PubMed1989

Monoclonal antibody directed against glutaraldehyde conjugated glutamate and immunocytochemical applications in the rat brain.

Like other small-sized neurotransmitter molecules, glutamate (Glu) was conjugated to carrier proteins via glutaraldehyde (G). Human serum albumin (HSA) and thyroglobulin (TH) conjugates were alternately injected into mice. When a relevant immune response was obtained for antibody affinity and specificity, hybridization of spleen activated lymphocytes with SP2/O/Ag myeloma cells was performed. Supernatant culture media of hybridomas were tested for the presence of anti-conjugated Glu antibodies with our ELISA method. Selected hybridomas giving good antibody affinity and specificity were then cloned by the limiting dilution technique. Using DEAE-chromatographed ascites fluid, Glu reactivity was observed on the cortex and the hippocampus. Staining obtained with this monoclonal antibody was in agreement with that observed with previous polyclonal antisera directed against conjugated Glu or monoclonal anti-gamma-glutamyl-Glu antibody.

Aldehydes↗

PubMed1989

Pre- and post-natal ontogeny of serotonergic projections to the rat spinal cord.

The development of 5-hydroxytryptamine (5-HT) innervation in the spinal cord was studied from embryonic day 14 (E14) to adulthood. Sprague-Dawley rats were fixed by perfusion with 5% glutaraldehyde in cacodylate-sodium metabisulfite buffer, and vibratome sections were processed for immunocytochemistry with a 5-HT antiserum. For electron microscopy, the sections were flat-embedded in araldite, and thin sectioning was performed. 5-HT neurons caudally directed from raphe nuclei invade the spinal cord at E14 and reach the caudalmost levels by E16-E17. In longitudinal sections, axons are seen by E15, at cervical and upper thoracic levels, to invade the presumptive gray matter from the anterior and lateral funiculi. The invasion process occurred either by sharp angulation of the axon or by branching of a collateral. By E16, at thoracic level the anterior horn and the intermediolateral columns are profusely innervated by very thin, varicose fibers; synapses are seen at E17 and E18 using EM. 5-HT immunoreactive boutons are involved here. After birth, 5-HT innervation of these two areas evolves progressively from a diffuse network to a more restricted pattern, especially at the thoracic level for the intermediolateral column and at cervical and lumbar levels for the anterior horn. The adult pattern is reached by postnatal day 21 (P21). The growth of axons toward the dorsal horn becomes noticeable by E19 at all spinal levels, when fibers invade the neck of the horn from the lateral funiculus, and innervation proceeds diffusely until P5. At P7, thin fibers course dorsally and laterally along the border of the gray matter and ramify profusely in layers I and II. The adult pattern is also reached in the dorsal horn by P21. These results are discussed in relation to the postnatal maturation of motor and sensory circuits and to the development of transplanted raphe neurons in the rat spinal cord.

Aging↗

PubMed1989

Identification of L-dopa-dopamine and L-dopa cell bodies in the rat mesencephalic dopaminergic cell systems.

An immunocytochemical technique for simultaneously visualizing two different antigens, dihydroxyphenylalanine (L-DOPA) and dopamine (DA), has been used to investigate the presence of cell bodies containing both compounds L-DOPA and DA and those having only L-DOPA in rat mesencephalon areas. The brain slices were processed with a double peroxidase-antiperoxidase method using simultaneously an incubation of a rabbit anti-L-DOPA serum and a monoclonal anti-DA antibody raised in mouse. Both antigens were revealed by the peroxidase reaction but with different chromogens that are easily distinguishable. In this staining procedure, the first antigen, conjugated DA was stained using the 3,3'-diaminobenzidine (DAB)-Nickel complex; while the second antigen, conjugated L-DOPA, was localized using DAB. The yellow-brown color due to DAB was masked by that of DAB-nickel. The possible existence of both single and double labelings could be worked. We have found many L-DOPA-positive/DA-positive and a few L-DOPA-positive/DA-negative cell bodies in dopaminergic regions in the rat midbrain: substantia nigra, ventral tegmental area, and raphe nuclei. In the locus coeruleus, we noted only L-DOPA-positive/DA-positive cell bodies. These results confirm those previously described for rat and cat hypothalamus, where both immunoreactive-cell body types have been detected: L-DOPA positive/DA positive and L-DOPA positive/DA negative. The existence of neuronal cells containing only L-DOPA is a new neuroanatomic finding, accounting better for the heterogeneity of dopamine systems with respect to physiologic, pharmacologic, and molecular data.

Animals↗

PubMed1989

Simultaneous immunogold labeling of GABAergic terminals and vasopressin-containing neurons in the rat paraventricular nucleus.

The GABAergic innervation of vasopressin-containing cells in the magnocellular part of the paraventricular nucleus was studied at the electron-microscope level using antibodies against GABA and vasopressin. The detection of both GABA and vasopressin on the same ultrathin section, performed with a double-labeling immunogold method, revealed GABAergic terminals in symmetrical synaptic contact with vasopressin-containing neurons. These GABAergic terminals displayed mitochondria, clear synaptic vesicles and varying numbers of electron-dense vesicles. Vasopressin-immunoreactivity was associated with neurosecretory granules, whereas GABA-immunoreactivity was found above mitochondria, clear synaptic vesicles and some electron-dense vesicles. This study, demonstrating the extensive participation of GABA in the innervation of magnocellular vasopressin-secreting neurons, suggests that this inhibitory neurotransmitter regulates vasopressin secretion at the level of the paraventricular nucleus.

Animals↗

PubMed1989

Host afferents into intrastriatal transplants of fetal ventral mesencephalon.

Host afferents into fetal ventral mesencephalic tissue grafted to the neostriatum of adult rats have been studied by using anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and immunocytochemistry for serotonin (5-HT), Substance P (SP), and dopamine-adenosine 3':5'-monophosphate-regulated-phosphoprotein-32 (DARPP-32). Numerous fibers of cortical origin were detected in the transplants following multiple (11-15) iontophoretic injections of PHA-L into the frontal and anterior cingulate cortex. The labeled fibers occurred with an apparently random distribution throughout the graft tissue. Their overall density was lower than that of the surrounding striatum but similar to that found in the host nigra-ventral tegmental area. The majority of the PHA-L-labeled fibers in the grafts were thin and tortuous with varicosities or lateral clubs with terminal boutons. Dual labeling showed frequent close appositions between PHA-L-labeled terminals and dopamine-immunoreactive cell bodies. In parallel electron microscopy, synaptic contacts were observed between PHA-L-labeled terminals and unlabeled neuronal profiles in the graft. Other labeled fibers in the grafts were thick and smooth, corresponding probably to labeled myelinated axons observed in the electron microscope. These thick fibers were often seen to give off collaterals of the thin type. The virtual absence of such thick fibers in the normal striatal neuropil suggests that at least some of the cortical afferents to the grafts may have sprouted from axons normally projecting to diencephalic or brain stem regions. Serotonin fibers occurred in patches or as scattered single fibers in both deep and superficial portions of the nigral transplants. In the electron microscope some of these terminals were seen to establish synaptic contacts with nonimmunoreactive elements in the graft. These fibers were present also when the graft tissue had been pretreated with 5,7-dihydroxytryptamine at the time of transplantation. This treatment eliminated all 5-HT-containing neurons from the grafts without any noticeable adverse effect on the survival of the dopaminergic neurons. The serotonin fibers in the grafts were thus most likely of host origin. SP-positive fibers formed a dense plexus inside the grafts. Since many SP-positive cell bodies were visualized inside the transplant after colchicine pretreatment, it is unclear, however, whether any of these fibers were of host origin. Intrastriatal injections of PHA-L or DARPP-32 immunocytochemistry indicated that the deep portions of the nigral grafts were entirely devoid of host striatal afferents.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗

PubMed1989

Identification and characterization of anti-conjugated azelaic acid antibodies in multiple sclerosis.

Human sera from patients with multiple sclerosis (MS) were tested using an enzyme-linked immunosorbent assay (ELISA) method on well plates coated with various dicarboxylic acid (C4 to C10) protein conjugates. Specific immunological binding was found with an azelaic acid (AzeA, C9) conjugate. The antibody titer was higher in the sera from the patients in acute relapse than with the progressive form, and higher than that from sera of patients with other neurological diseases and healthy subjects. Modifications of coating concentrations and of antibody dilutions, and experiments with preadsorption enabled determination of binding specificity. Competition experiments with related conjugates demonstrated that the AzeA residue was 167 times better recognized by antibodies from MS patients in acute relapse than those from controls. The suberic and sebasic acid conjugates which only differ from the AzeA conjugate by one methylene group were less well-recognized by MS sera (11 and 47 times, respectively) than the conjugate AzeA-BSA.

Antibodies↗

PubMed1989

Central dopamine-synthesis regulation by the calcium-calmodulin-dependent system.

The effects of the intraventricular (IVT) administration of calcium on the amount of dopamine (DA) in various regions of the mouse brain were analyzed immunohistochemically by using a microphotometry system. The DA levels in the nucleus accumbens and the lateral part of the neostriatum were increased by approximately 45% (p less than 0.01) and 25-35% (p less than 0.01), respectively, by the IVT administration of CaCl2 (10 mumol/kg). It was also found that this effect was abolished by the calmodulin antagonist, W-7 (4.2 micrograms/mouse, IVT). The brain regions in which the amount of DA was increased by calcium were areas where high levels of calmodulin and tyrosine hydroxylase are distributed. These findings suggest that the synthesis of central DA is regulated by calcium through a calmodulin-dependent system.

Animals↗

PubMed1989

Intraspinal transplants of serotonergic neurons in the adult rat.

Adult male Sprague-Dawley rats were made paraplegic by a complete transection of the spinal cord at lower thoracic level. One week later they were transplanted, below the level of the section, with a cell suspension prepared from the raphe region of 14-day embryos. After survival periods of 10 days to 1 year, the animals were sacrificed and the spinal cord processed for the immunocytochemical detection of 5-HT. Axons from grafted cells grew extensively into the grey matter of the host, and established axosomatic and axodendritic synapses in the anterior horn and intermediolateral column, similar to those of the intact animal. In addition, a group of transplanted animals was tested for sexual reflexes which are under the control of serotonin. It was found that ejaculation reflexes, which are absent in paraplegic rats, are restored in transplanted animals.

Animals↗

PubMed1989

Morphological and electrophysiological studies of human hippocampal transplants in the anterior eye chamber of athymic nude rats.

Human fetal hippocampal tissue from normal women was obtained following elective abortion in the 8th to the 11th week of gestation. The hippocampal tissue was transplanted to the anterior chamber of the eye of adult athymic nude rats, where it was allowed to develop for up to 9 months before histological and electrophysiological evaluation. The transplants were revascularized from the host iris and many grew extensively in oculo. Large neurons were present in all transplants. Immunohistochemical studies revealed glutamic acid decarboxylase-containing terminals and clusters of gamma-aminobutyric acid-positive nerve cell bodies within the transplants, as well as scattered tyrosine hydroxylase-positive and acetylcholinesterase-containing fibers. Single neurons recorded extracellularly from transplants 4-9 months in oculo showed a slow spontaneous discharge, with both complex and single action potentials. Stimulation of the transplant surface evoked a small initial wave followed by a larger and longer-lasting field potential, similar to that seen in hippocampus in situ. A conditioning-testing paradigm was used to evaluate the presence of inhibitory circuitry in the hippocampal transplants. Significant suppression of the evoked test response was seen with interstimulus intervals ranging from 20 to 500 ms. Superfusion of enkephalin (100-300 nM) or penicillin (1600 U/ml) increased slow-wave activity, as did tetanic electrical stimulation. These treatments appeared to generate ictal-like activity, which in some cases persisted as interictal spikes. Illumination of the retina also increased neuronal activity, presumably by reflex activation of cholinergic afferents from the parasympathetic innervation of the iris. Taken together, our data suggest that fragments of hippocampus from aborted first trimester human fetuses, grafted to the eye chamber of rodent hosts, develop many organotypic histological and physiological features. This preparation may provide a unique means for the study of neurobiological properties of human brain in both normal and disease states.

Animals↗

PubMed1989

Dopamine synaptic complex with pyramidal neurons in primate cerebral cortex.

Dopamine (DA)-containing projections to the cerebral cortex are considered to play an important role in cognitive processes. Using a recently developed monoclonal antiserum directed against DA and an antibody directed against tyrosine hydroxylase in combination with Golgi impregnation and electron microscopy, we have observed that DA and tyrosine hydroxylase afferents establish symmetric membrane specializations with the soma, dendritic shafts, and spines of identified pyramidal cells in the prefrontal, cingulate, and motor cortex of primates. The axospinous contacts invariably formed part of a synaptic complex in which the dendritic spine of a pyramidal neuron was the target of both a DA-positive symmetric and an unlabeled asymmetric bouton. This arrangement allows direct DA modulation of the overall excitability of cortical projection neurons by altering local spine responses to excitatory inputs.

Animals↗

PubMed1989

Monoclonal anti-conjugated acetylcholine antibody and immunohistochemical applications in rat nervous system.

Acetylcholine (ACh) conjugates were injected into AKR and DBA mice over a period of 10 weeks. The polyclonal antisera were tested at various immunization times for affinity and specificity using an enzyme-linked immunosorbent assay (ELISA). The most immunoreactive compound was found to be choline-glutaryl-bovine serum albumin (or conjugated ACh). The AKR and DBA mice yielding the highest apparent affinity were killed, and the spleen cells were fused with X63 or SP2/O/Ag mouse myeloma cells. Supernatants of confluent cultures were tested for the presence of anti-conjugated ACh antibodies using the same ELISA method. The best results were obtained with the hybridomas from AKR spleen cells and X63 mouse myeloma cells. Monoclonal antibody affinity and specificity were then evaluated by a radioimmunological procedure using iodinated monoclonal anti-conjugated ACh antibody. From competition experiments, the most immunoreactive compound was choline-glutaryl-protein. The other related compounds were recognized either poorly or not at all. The high affinity and specificity of our monoclonal antibody enabled us to visualize ACh molecules on fixed rat brain sections. ACh was fixed with a mixture of nitrobenzyl alcohol and glutaraldehyde. Many ACh-immunoreactive cell bodies and fibers were seen on sections from the basal forebrain and spinal cord. Preadsorption and other immunohistochemical tests demonstrated that the ACh staining was highly specific.

Acetylcholine↗

PubMed1989

Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects.

Previous immunohistochemical studies of the long-term effects of the noradrenergic neurotoxin DSP-4 have demonstrated a remarkably selective vulnerability of norepinephrine (NE) axons of the locus coeruleus (LC). NE axons originating in non-LC NE neurons appear to be largely resistant to the neurotoxic action of DSP-4. We conducted this study to evaluate the acute effects of DSP-4 on NE axons in four different brain regions: cerebral cortex, cerebellum, ventral forebrain, and hypothalamus. NE levels were determined by high-performance liquid chromatography (HPLC) 6 and 24 hr and 14 days after DSP-4 administration. NE axons in these brain regions were visualized in brain sections at 6 and 24 hr after drug treatment, using a specific antiserum to NE. HPLC assays revealed profound reductions of NE levels in cerebral cortex and cerebellum, but only minor decreases in ventral forebrain and hypothalamus. NE immunohistochemistry showed dramatic differences in the acute effects of DSP-4 on NE axon staining: nearly complete loss of staining in cortex and cerebellum, in contrast to an almost unchanged staining pattern in ventral forebrain and hypothalamus. This study demonstrates that NE immunohistochemistry is a valuable tool to assess the acute effects of DSP-4 on NE axons in different brain regions. The results provide the first direct evidence that NE axons are not uniformly acted on by DSP-4 and suggest that the acute effects of DSP-4 are restricted to LC axons.

Animals↗

PubMed1989

[Cells presenting GABA immunoreactivity in the hypothalamus of the cat].

The distribution of GABA-immunoreactive (IR) cells was studied by immunohistochemistry in conjunction with highly specific antiserum GABA in the cat hypothalamus. Colchicine pretreatment made it possible to visualize a large number of labeled cells in the medial preoptic and dorsal hypothalamic areas. In contrast, the ventromedial and anterior hypothalamic nuclei contained only a few labeled cells, and the paraventricular and supraoptic nuclei were devoid of them. A very dense network of GABA-IR presumptive terminals was seen in the ventrolateral posterior hypothalamus where labeled cells could be recognized. The possibility of an involvement of the GABAergic neuronal system in the regulation of sleep-waking cycle is discussed.

Animals↗

PubMed1989

[Autoantibodies directed against membrane phospholipid in serum in patients with malignant tumors].

Modifications of membrane lipids in the levels of fatty acids and phospholipids are associated with malignant tumors. In order to evaluate if these changes are recognized by the immune system, we have attempted to assay the possible presence of autoantibodies directed against the following lipids: phosphatidylinositol (PI), phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, gangliosides, galactocerebrosides, sphingomyelin, sphingosin, and cardiolipin, in the sera of patients with malignant tumors (n = 324) and from controls [healthy subjects (n = 20) and patients suffering of other diseases (n = 60)]. Using an adaptated immunoenzymatic assay (ELISA method), a highly significant difference (p less than 0.001) was found between the mean absorbances read on the cancer and control group for only one lipid, the PI. Whatsmore, these auto anti-PI were found in all sera (diluted 15,000 times) of patients with malignant tumors, whatever type, grade, or organ localization defined. These data indicated that the immune system recognized the PI antigenic modifications which appear to be linked to the cell transformation. This PI-immunological binding may have a predictive value as we have recently noticed in animals bearing chemically-induced malignant tumors.

Autoantibodies↗

PubMed1989

Critical review on quantitative autoradiography of D1 and D2 dopaminergic receptors in the striatum of the mammalian brain: differential localization and plastic changes after pharmacological manipulation and dopaminergic input disruption.

Major technical progress in the development of computer-based image analysis systems has made possible the entry of autoradiographic and immunohistochemical techniques into a new era where quantification via densitometry and morphometry has become easily accessible. In this context, quantitative biochemical data can be adapted to anatomical and histological resolution. This adaptation is most efficient in the neuroscience fields because of the huge importance of cellular communication via neuronal networks in the nervous system. Therefore, any experimental approach to the brain which considers the brain as a 'black box' appears now as very crude. In fact, subtle heterogeneity in the distribution of biochemical markers can now be demonstrated, as illustrated here by the use of quantitative autoradiography of D1 and D2 dopaminergic receptors in the striatum of the mammalian brain. Also, local adaptive changes resulting from chronic blockade of the dopaminergic input can be detected after repeated treatments with dopaminergic antagonists selective for D1 or D2 receptors or with surgical lesioning of the dopaminergic nigrostriatal pathway. The resulting plastic changes are unevenly distributed throughout the striatal target organ and vary according to the mode of suppressing the dopaminergic flow: direct destruction of the dopaminergic pathway or selective pharmacological manipulation without physical elimination of the dopaminergic cells themselves. All these results are discussed and reviewed in light of the most recent reports in this field.

Animals↗