Biomedical subjects

M Geffard

Publications and source records attributed to M Geffard.

At least 109 records · Page 6Linked to original sources

PubMed1990

Noradrenaline axon terminals in adult rat neocortex: an immunocytochemical analysis in serial thin sections.

Peroxidase-antiperoxidase electron microscope immunocytochemistry with an antiserum against noradrenaline-glutaraldehyde-protein conjugate was used to identify cortical noradrenaline terminals (axonal varicosities) from the upper layers of the frontal, parietal and occipital cortex in adult rat. A large number of immunostained varicosities were examined in serial thin sections, and compared with a control population of randomly chosen unlabeled terminals from the same sections. Both groups of varicosities were measured and scrutinized for the presence of a junctional complex indicative of synaptic specialization. Cellular elements juxtaposed to the membrane of both types of varicosities were also identified and counted. Noradrenaline varicosities in all three cortical regions averaged 0.65 microns in diameter. In contrast to their unlabeled counterparts, these profiles rarely showed a membrane differentiation characteristic of a synaptic contact (junctional complex). The rare junctional complexes formed by cortical noradrenaline varicosities were invariably symmetrical and almost always found on dendritic shafts. The microenvironment of noradrenaline varicosities also differed, exhibiting a greater number of apposed axonal varicosities and a smaller number of dendritic spines than that of the random population. The proportion of noradrenaline varicosities making a synaptic contact (synaptic incidence) was determined by plotting the incidence of visible junctions as a function of the number of thin sections available for examination. As extrapolated for whole varicosities after linear transformation (double reciprocal plot), this proportion was 17% or 26% depending on the stringency of the criteria used in identifying the junctional complex. The same analysis provided a figure of 98% for the control population. The present study largely confirmed our initial radioautographic characterization of the cortical noradrenaline innervation as a mostly non-junctional system, and also indicated that these varicosities are set in a particular microenvironment. These new data further support the eventuality of a diffuse release of cortical noradrenaline in the extracellular space, compatible with both its neuromodulatory role and multiplicity of actions on diverse cellular targets in the cerebral cortex. The functions assigned to the coeruleocortical noradrenaline system must therefore be viewed as the product of a widespread and ubiquitously distributed neuronal organization characterized by loose intercellular relationships. This system might be capable of selectivity and specificity of action, however, owing to the distribution of its receptors, and in view of intrinsically or extrinsically driven control mechanisms triggered by the release of its own or other transmitters and which may also involve target-initiated feedback mechanisms.

Animals↗

PubMed1990

Neurons containing messenger RNA encoding glutamate decarboxylase in rat hypothalamus demonstrated by in situ hybridization, with special emphasis on cell groups in medial preoptic area, anterior hypothalamic area and dorsomedial hypothalamic nucleus.

Previous deafferentation studies have suggested that most hypothalamic GABAergic innervation originates from neurons within the hypothalamus. We have investigated the distribution of GABAergic cell groups in the rat hypothalamus by means of the in situ hybridization technique, using a cDNA probe for messenger RNA encoding glutamate decarboxylase. Several major GABAergic cell groups were demonstrated, including cells of the tuberomammillary nucleus, arcuate nucleus, suprachiasmatic nucleus, medial preoptic area, anterior hypothalamic area, the dorsomedial hypothalamic nucleus, perifornical area, and lateral hypothalamic area. The most prominent glutamate decarboxylase mRNA-containing cell groups were located in the medial preoptic area, anterior hypothalamic area and dorsomedial hypothalamic nucleus, and were composed of small- to medium-sized neurons. Compared to previously well-characterized GABAergic cell groups in the tuberomammillary nucleus, reticular thalamic nucleus, and non-pyramidal cells of cerebral cortex, the cells of these GABAergic groups demonstrated only weak cDNA labelling, indicating that they contain lower levels of glutamate decarboxylase mRNA. Several types of control experiments supported the specificity of this cDNA labelling, and the GABAergic nature of these cell populations was further supported by detection of glutamate decarboxylase and GABA immunoreactivity. Abundance of GABAergic cells in many hypothalamic nuclei indicates that GABA represents quantitatively the most important transmitter of hypothalamic neurons, and may be involved in neuroendocrine and autonomic regulatory functions.

Animals↗

PubMed1990

Dissimilar responses of adult thalamic monoaminergic and somatosensory afferent fibers to implantation of thalamic fetal cells.

It is generally accepted that transplanted fetal neurons can, after several weeks to months, establish connections with the host CNS. Host afferent systems seem, however, to show different types of responses to the presence of grafted fetal neurons. The present study is a preliminary step to identify mechanisms involved in the reactions of adult axons to transplanted fetal neurons. The right ventrobasal thalamus of adult rats was depleted of neurons by in-situ injection of kainic acid and cell suspensions from homotopic thalamic embryonic primordia which were injected into the lesioned area. After various post-implantation delays, ranging from five to 30 days, two types of experiments were performed: (i) noradrenaline and serotonin immunohistochemistry with specific antibodies on alternate sections; and (ii) anterograde tracing using wheat germ agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei and the principal sensory trigeminal nucleus. Five days after transplantation, host monoaminergic fibers (either noradrenergic or serotoninergic) had already grown into the transplants. Ingrowing fibers were thin and poorly varicose, exhibiting endings morphologically similar to the growth cones observed during axogenesis. Seven days after grafting, growth cones were no longer visible and monoaminergic fibers exhibited either normal-sized or very large varicosities. Large varicosities progressively decreased in number and, after three weeks, the fibers displayed a normal adult morphology, forming a dense network all over the transplants. In contrast, host somatosensory afferents, labeled by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, did not grow into the transplants. Intermingling of somatosensory afferents and transplanted cells was observed only after 10 days, when grafted neurons extended outside the original transplantation site into the neuron-depleted area containing the somatosensory afferents. The present results demonstrate that adult monoaminergic and somatosensory afferents, when deprived of their usual target, do not react in a similar way to the addition of fetal neurons. It is proposed that adult monaminergic fibers have the ability to regain morphological (and probably functional) immature forms which were considered to be restricted to the period of axogenesis or to lesion-induced regeneration. In contrast, fetal transplants do not seem to induce, by themselves, a similar alteration of genetic expression in adult somatosensory neurons. It has been proposed that "diffuse" and "point-to-point" axonal systems may be differentiated in the CNS on anatomical bases. The present results add to the identification of two different systems by demonstrating that, in the thalamus, they present dissimilar responses to the implantation of fetal cells.

Animals↗

PubMed1990

Monoclonal anti-conjugated azelaic acid antibody production: application to multiple sclerosis.

We have previously reported the existence of anti-conjugated azelaic acid (Aze A) antibodies in the serum of patients with multiple sclerosis (MS). In order to demonstrate the specificity of these antibodies, we have produced a monoclonal antibody directed against Aze A conjugated by an acylation reaction to a protein. In competition experiments, with ELISA method, we demonstrated that a part of the antibodies, raised in rabbit after immunization by human immunoglobulins (Ig) of MS patients, recognized the antigen-combining site of our monoclonal anti-conjugated Aze A antibody. These results clearly demonstrate that a part of human Ig obtained from sera of MS patients shared common idiotopes with mouse monoclonal antibody raised against conjugated Aze A.

Acylation↗

PubMed1990

Existence of L-dopa immunoreactive neurons in the rat preoptic area and anterior hypothalamus.

We demonstrate the presence of L-DOPA (L-3,4-dihydroxyphenylalanine)-labelled cell bodies and fibers in the rat preoptic and anterior hypothalamic areas, using a rabbit polyclonal antiserum against conjugated L-DOPA and a glutaraldehyde coupled immunohistochemical technique. In contrast, using a monoclonal anti conjugated dopamine (DA) antibody, no DA labelled neurons were detectable in the preoptic region, whereas a few weakly immunostained DA neurons began to appear in the ventral part of the medial preoptic area. These observations point to the hypothesis that these cells contain mainly L-DOPA neurons in the preoptic and anterior hypothalamic areas. Thus, we suggest that L-DOPA plays a more important role in the hypothalamic functions than has been hitherto assumed.

Animals↗

PubMed1990

The distribution of 5-HT immunoreactive systems in the brain of a saurian, the chameleon.

The distribution of serotonin immunoreactive cell bodies and fibers was studied in the chameleon brain by using the immunohistochemical technique with antisera against serotonin coupled to a carrier with glutaraldehyde. Serotonin perikarya were found in the caudal midbrain tegmentum, in the lateral part of the nucleus reticularis isthmi, the lateral part of the nucleus interpeduncularis and along the midline in the raphe superior. More caudally, the serotonin immunoreactive cell bodies were located along the nucleus raphe inferior and ventrolaterally in the vicinity of the olivary complex. No immunoreactive cell bodies were found in the spinal cord nor in the paraventricular organ (PVO) of the hypothalamus. Immunoreactive fibers were observed in the entire brain. Prominent concentrations were found in the dorsal cortex, lateral septum, lateral geniculate nucleus, median eminence, pretectal nucleus, nucleus interpeduncularis, vestibular nucleus and olivary complex. Descending serotonin immunoreactive fibers were found in particular in the ventral motoneuron area in the spinal cord. One of the most interesting findings in this study was the lack of immunoreactive CSF contacting neurons in the PVO and the observation of an extensive plexus of supraependymal fibers, a feature reported so far only in mammals.

Animals↗

PubMed1989

Co-localization of tyrosine hydroxylase and GABA immunoreactivities in human cortical neurons.

Samples of human cerebral cortex were stained immunocytochemically for tyrosine hydroxylase (TH) and gamma-aminobutyric acid (GABA). TH-positive neurons were in small number and predominated in the deep infragranular layers V-VI contrasting with numerous GABA-positive neurons scattered in all layers. Co-localization of TH- and GABA-like immunoreactivities in a single cell was studied by the double immunolabeling technique with the elution-restaining procedure. Only 50% of the TH-positive neurons also expressed GABA-like immunoreactivities. The two markers were detectable in the somata and not in the processes of the cells. The double-labeled cells were mainly fusiform and medium-sized and were observed in layer VI. These observations suggest that the TH-positive cells form a mixed neuronal population, only a part of which corresponds to the GABAergic class of intrinsic interneurons.

Cerebral Cortex↗

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↗