Biomedical subjects

S Araneda

Publications and source records attributed to S Araneda.

29 records · Page 2Linked to original sources

PubMed1991

[Gaba innervation of serotonergic neurones innervating the thoracic marrow of the rat. Immunocytochemical and autoradiographic combination after retrograde axonal transport with tritiated serotonin ].

Anatomical relationship between GABA terminals and serotonergic neurons that innervate the thoracic spinal cord was studied using combined methods: autoradiography following (3H) 5-HT retrograde transport and immunocytochemistry for GABA. Following (3H) 5-HT injections into the thoracic spinal cord, serotonergic neurons of raphe pallidus and magnus were specifically radiolabelled. At optic and electron microscopic levels, radiolabelled cells of raphe magnus and pallidus were in contact with GABA immunoreactive pre-synaptic nerve terminals. Some radiolabelled cell bodies were contacted by both GABA immunoreactive and immunonegative terminals. These results indicate the existence of a direct GABAergic input onto the medullary serotonergic neurons projecting to the thoracic spinal cord and suggest both GABAergic and non-GABAergic regulation of these serotonergic neurons. Thus, combination of both a specific tracing technique and immunocytochemistry offers considerable advantages of specificity and sensibility for the study of interrelationship between chemically defined neuronal pathways and/or of transmitter co-existence.

Animals↗

PubMed1989

Tracing specific transmitter pathways in the rat CNS: combination of [3H]serotonin retrograde labelling with immunocytochemical detection of endogenous transmitters.

Selective retrograde labelling with [3H]serotonin ([3H]5-HT) can be used to identify serotonergic cell bodies after specific [3H]5-HT uptake by the corresponding nerve terminals. In the present study, we demonstrate that autoradiography of this [3H]5-HT radiolabelling can be combined with immunocytochemical detection of endogenous serotonin, GABA or substance P on the same tissue section. The midbrain raphe serotonergic projections to the olfactory bulb and the spinal projections of medullary serotonergic nuclei were investigated. The specificity of retrograde labelling with [3H]5-HT was confirmed by immunoreactivity of the radiolabelled cells for serotonin, using an antiserum specific for formaldehyde-fixed serotonin. After spinal injections of [3H]5-HT, many retrogradely labelled cells in the medullary raphe were immunopositive for substance P, and a few for GABA. These results are in agreement with the available information on the co-existence of putative transmitters in the spinal projections of caudal raphe neurons. Therefore, autoradiography of [3H]5-HT retrograde labelling combined with immunocytochemistry offers a possibility to test the specificity of transmitter-selective retrograde labelling, to identify transmitter-defined neuronal interactions and to investigate the projection fields of multitransmitter containing neurons.

Animals↗

PubMed1989

[3H]-serotonin retrograde labelling in serotonergic fibers.

Autoradiography, following retrograde axonal transport with [3H]-serotonin (10(-4) M) was used to identify, selectively, the serotonergic neurons of medullary raphe that innervate the spinal cord. In combination with this technique, immunocytochemical detection of endogenous serotonin showed some raphe magnus cell bodies immunostained by 5-HT antibodies and [3H]5-HT radiolabelled. Serotonergic fibers of lateral and dorsolateral funiculus of ligated spinal cord were also characterized following retrograde axonal transport with 10(-4) M [3H]-serotonin. At the ultrastructural level, mainly myelinated, although unmyelinated labelled fibers were also identified as swollen axons. Clusters of large membranous organelles, predominantly mitochondria, some dense lamellar bodies, multivesicular bodies and lysosomes accumulated on the distal segment of the ligation. Autoradiographic reaction was always observed to be very intense and silver grains overlapped mainly mitochondria (41%) and clusters of membranous organelles (37%). However, smooth endoplasmic reticulum seemed to be devoid of silver grains. From our results, serotonergic myelinated spinal fibers were labelled after uptake-retrograde axonal transport with [3H]5-HT. Mitochondria and membrane organelles could convey tritiated compounds derived from [3H]-serotonin uptake towards the serotonergic cell bodies.

Animals↗

PubMed1988

Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP.

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.

Adenosine Triphosphate↗

PubMed1988

Serotonergic projections to the spinal cord but not those to the olfactory bulb also contain substance P. A combined immunocytochemical and autoradiographic study following retrograde axonal transport of [3H]serotonin labeled products.

Co-localization of substance P with serotonin in raphe projection neurons was studied by combining substance P immunocytochemistry and autoradiography following uptake and retrograde axonal transport of [3H]serotonin and/or its products from target areas. In this study, two central pathways in the rat were investigated: the serotonergic projections of the midbrain raphe to the olfactory bulb and those of the medullary raphe that innervate the thoracic spinal cord. Two hours after pargyline pretreatment, injections of 10(-4) M [3H]serotonin were made either into the olfactory bulb or into the spinal cord and respectively 24 or 60 h thereafter, rats were administered with colchicine. After a 24 h survival time, the paraformaldehyde fixed brains were investigated for substance P immunocytochemistry and then treated for light and electron microscopy autoradiography. Combining both methods, we can define on the same tissue sections at least three labeled neuronal populations: substance P immunolabeled neurons, radiolabeled neurons and doubly immuno-radiolabeled neurons. In the midbrain raphe cells as well as in the olfactory bulb nerve terminals, two kinds of labeled profiles were detected: substance P immunoreactive profiles and radiolabeled ones. The radiolabeled cell bodies of the midbrain raphe (403 counted cells) were never reactive to substance P antibodies. Moreover, they were distributed caudally to substance P stained perikarya. In contrast, in the medullary raphe, of the 336 radiolabeled cell bodies 162 were stained after substance P antibody treatment. They represent about 48% of the serotonin radiolabeled neurons projecting to the thoracic spinal cord, where a great number of varicosities were observed immunolabeled, radiolabeled and doubly immuno-radiolabeled in the dorsal horn. At the ultrastructural level, cell bodies and dendritic processes were also doubly labeled. Both labelings were observed over the cytoplasm and some organelles or perikarya. These observations provide a morphological basis to support the hypothesis that substance P can occur within some but not all serotonergic neurons and raise questions about the expression of this peptide in these systems as well as the modes of interaction of these transmitter molecules.

Animals↗

PubMed1984

Effect of local 5,6-dihydroxytryptamine on the rat olfactory bulb responsiveness during wakefulness and sleep.

This study investigated a possible participation of the centrifugal serotonergic (5-HT) innervation of the olfactory bulb (OB) in the control of the multiunit mitral cell responsiveness in behaving rats. A bilateral injection of 5,6-dihydroxytryptamine solution (5,6-DHT) into the OB induced in this structure an average 70% reduction in the level of endogenous serotonin. Control and treated animals were habituated to eat a single 2 h daily meal. Recording sessions were performed either after a 22 h fasting period or just after the meal. The summated multiunit mitral cell activity and vigilance state parameters were recorded in the absence of and during repeated stimulation by puffs of food odour and isoamyl acetate. The results showed that the 5,6-DHT treatment did not modify significantly previously established centrifugal controls exerted at the OB level: (1) the selective enhancement of the mitral cell responsiveness to food odour occurring in waking food-deprived animals; (2) the inhibition of the olfactory input during slow wave sleep. However, the considered 5-HT projections could mediate some inhibitory influence on mitral cell activity during wakefulness only. Finally, the 5-HT innervation of the OB did not seem to be involved in basic mechanisms of olfactory recognition.

5,6-Dihydroxytryptamine↗

PubMed1983

Effect of local 6-OHDA and 5,6-DHT injections into the rat olfactory bulb on neophobia and learned aversion to a novel food.

This study investigated the role of the noradrenergic (NA) and the serotoninergic (5-HT) centrifugal fibers on the olfactory bulb (OB). For this purpose, behavioral responses largely depending on olfactory information were measured in rats after discrete bilateral injections into the OB with either 6-hydroxydopamine (6-OHDA) or 5,6-dihydroxytryptamine (5,6-DHT) solutions. The evaluation of the level of endogenous 5-HT in the OB was measured by high performance liquid chromatography. The NA fibers appeared lesioned after the 6-OHDA treatment only, whereas the 5-HT level was significantly reduced in 5,6-DHT treated bulbs only (34% of the control value). The animals were fed on usual stock-diet (S) and had the choice between S and a novel food (V) on neophobia test, and again on the following day after aversive conditioning (0.9 mEq/kg LiCl i.p.). The behavioral results showed that the drug administrations were followed by a significant effect in one case only: the intake of V was significantly enhanced in 6-OHDA treated animals during neophobia test. Thus, neophobia was reduced by 6-OHDA. The data are interpreted as follows: (1) the lesion of the bulbar NA or 5-HT fibers does not induce total anosmia; and (2) the NA innervation of the OB could play a role in the modulation of behavioral responses to biological odorants.

5,6-Dihydroxytryptamine↗

PubMed1982

Selective retrograde transport of 3H-serotonin in vagal afferents.

A new serotonergic afferent vagal component has been demonstrated in the cat by radioautography. Twenty-four hours after a bilateral injection of tritiated serotonin (3H-5-HT) into the area of the nucleus of the solitary tract (NST), heavily and lightly labelled cell bodies were observed in the nodose ganglia. After unilateral injections of 3H-5-HT into the same area, labelled ganglionar cell bodies were found in the ipsilateral nodose ganglion. Some were also found in the contralateral one, suggesting a serotonergic crossed fibers component. Dense clusters of silver grains, depicting typical labelling of neuronal varicosities, were observed in the NST. After destruction of the serotonergic terminals with 5,7-dihydroxytryptamine, followed by injection of 3H-5-HT, the number of labelled cell bodies decreased dramatically in the ipsilateral nodose ganglia and the clusters of silver grains disappeared in the NST. After ligature or section of the supranodose vagal nerve, following injection of 3H-5-HT into the NST, no radioautographic reaction was observed in the homolateral nodose ganglia. The present study demonstrates the existence of a peripheral serotonergic system in vagal afferents. The physiological implications of this new serotonergic visceral pathway remain to be studied.

Afferent Pathways↗

PubMed1981

Is the retrograde axonal transport of 3H-5-HT a specific process of serotoninergic neurons?

The specificity of the retrograde axonal transport of 3H-serotonin (3H-5-HT) was radioautographically studied in the afferents to the olfactory bulb (O.B.). Injections of 3H-5-HT of different concentrations (10(-2), 10(-3), 10(-4) and 10(-5) M) were performed into the O.B. of catron pretreated rats. Following injection of 3H-5-HT (10(-2) M), a cytoplasmic perikaryal labeling was observed in the bulk of afferents to the O.B. (aminergic and non-aminergic neurons). When lower concentrations of 3H-5-HT (10(-5) M) were injected into the O.B., the retrograde labeling was only seen in the raphe dorsalis (RD) serotoninergic perikarya. The specificity of the uptake-retrograde transport of 3H-5-HT seems to depend on the selectivity of uptake by nerve terminals.

Afferent Pathways↗

PubMed1980

Retrograde axonal transport following injection of [3H]serotonin in the olfactory bulb. I. Biochemical study.

A retrograde axonal transport from the serotonergic nerve terminals in the olfactory bulb (OB) to their parent cell bodies in the midbrain raphe nuclei has been demonstrated after stereotaxic injection of [2H]5-HT into the OB of rats pretreated with a monoamine oxidase (MAO) inhibitor: at various time intervals thereafter (4-92 h) there was a preferential accumulation of radioactivity mainly in the raphe dorsalis nucleus (RDN). Maximal accumulation occurred at 24 h. Of this radioactivity, 30-50% was recovered as 5-HT. The accumulation was estimated to take place at two rates: a fast one (48 mm/day) and a slower one (16 mm/day). Under the same experimental conditions there was no clear evidence for a retrograde accumulation of [3H]norepinephrine in the RDN. A passive diffusion mechanism could be excluded since the diffuson of tracer towards the cerebrospinal fluid was prevented by prior mechanical obstruction of the olfactory diverticle of the lateral ventricle. Furthermore, colchicine strongly reduced (by 80%) the radioactive accumulatin in the RDN. Destruction of serotonergic nerve terminals by 5,6-dihydroxytryptamine or inhibiton of 5-HT uptake by fluoxetine decreased this retrograde accumulation whereas destruction of catecholaminergic nerve terminals by 6-hydroxydopamine was without effect. Pretreatment with reserpine decreased the amount of radioactivity transported to the RDN by 40%. In the absence of MAO inhibition pretreatment, animals still presentd 35% of the tracer transported to the RDN. Intrabulbar injection of MAO inhibitor did not affect the accumulation rates when compared with animals which received the inhibitor by the intraperitoneal route. In conclusion, the retrograde axonal transport following [3H]5-HT injection in the serotonergic RDN-OB system occurs via an active process which depends on a colchicine-sensitive mechanism and is partially linked to a reserp ine-sensitive structure. During its transport, the amine seems to be relatively protected from metabolic inactivation.

Animals↗

PubMed1980

Retrograde axonal transport following injection of [3H]-serotonin into the olfactory bulb. II. Radioautographic study.

Radioautography was used to study the intraneuronal distribution of [3H]-serotonin (5-HT) and/or its derivatives selectively taken up by the olfactory bulb (OB) serotonergic terminals and subsequently transported to their parent cell bodies in the midbrain raphe nuclei. This was done 24 h after injection of [3H]5-HT into the main OB of rats either pretreated or not with monoamine oxidase (MAO) inhibitor. A prior mechanical obstruction of the rostral ventricular cavities prevented diffusion of the tracer towards cerebrospinal fluid. Heavily labelled nerve cell bodies were found mainly in the ipsilateral raphe dorsalis nucleus (RDN) and to a lesser extent in the raphe centralis nucleus. The radioautographic reaction often extended to dendritic processes while sparing the nucleus. A diffuse reaction was also observed but limited to the raphe area. The supraependymal 5-HT fibers were found to be free of labelling. Neither local destruction of catecholaminergic terminals with 6-OHDA, nor absence of MAO inhibition, impaired this radioautographic pattern, while destruction of serotonergic terminals with 5,6-dihydroxytryptamine in OB resulted in the disappearance of labelled axonal varicosities and neurons in the OB and the RDN respectively. At the electron microscopy level, labelled cell bodies in the RDN were medium-sized (12-15 micrometers). Silver grains were localized mainly on mitochondria and, to a lesser extent, on lysosomes and endoplasmic reticulum but spared the nucleus and the nucleolus. Silver grains were also found near the nuclear membrane and outside the neuronal membrane. The observation of heavy metal impregnated thick sections confirmed the preferential localization of silver grains on mitochondria with or without inhibition of MAO. These results could account for the subcellular compartments involved in the retrograde axonal transport of [3H]5-HT and its subsequent degradation and/or dendritic release.

Animals↗