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J F Pujol

Publications and source records attributed to J F Pujol.

At least 91 records · Page 5Linked to original sources

Immunohistochemical evidence for the presence of gamma-aminobutyric acid and serotonin in one nerve cell. A study on the raphe nuclei of the rat using antibodies to glutamate decarboxylase and serotonin.

A specific and sensitive double immunocytochemical staining for the visualization of glutamate decarboxylase (GAD) and serotonin (5-HT) on the same brain section is developed. GAD is detected with specific GAD-antibodies by means of the unlabeled antibody enzyme, peroxidase anti-peroxidase, and serotonin with an antibody against the BSA-serotonin conjugate by an indirect immunofluorescent staining. The coexistence of GAD and 5-HT in the same perikaryon is demonstrated by a peroxidase reaction superimposed on fluorescent compounds. Cell bodies containing both antigens are observed in each raphe nuclei. However, the nucleus raphe dorsalis exhibits the largest number of cells containing either GAD alone or GAD and 5-HT together. An intracellular interaction between the metabolism of GABA and serotonin could be reasonably expected. The interactions between GABAergic and serotonergic systems must be thought of in terms of intracellular and/or transynaptic controls.

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Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. I. Nature and origin of contributors to the oxidation current at +0.1 V.

Differential pulse voltammetry was used together with treated carbon fiber microelectrodes to study the in vivo catecholamine (CA) metabolism in the locus coeruleus (LC), a brain region densely packed with noradrenergic neurons. In chronically implanted rats, an in vivo oxidation current that peaks at +0.1 V has been detected inside the LC complex. This current whose potential is characteristic of the oxidation of the catechols, had the same anatomical localization as the noradrenergic cells. Pharmacological experiments have been made to ascertain which catechols contribute to this in vivo current. Monoamine oxidase inhibition by pargyline was followed by a total and rapid suppression of the in vivo signal. Blockade of dopamine-beta-hydroxylase by FLA-63 induced a significant increase in the electrochemical signal. Post-mortem analysis of LC catechol levels after administration of this drug revealed a considerable decrease in NA and its major catechol metabolite, 3,4-dihydroxyphenylglycol (DOPEG) although DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were significantly increased. Comparison of these results led us to conclude that DOPAC is probably the most important contributor to the in vivo oxidation current. This assertion is corroborated by results obtained after tyrosine hydroxylase inhibition with alpha-methyl-p-tyrosine: the in vivo catechol current was rapidly suppressed and post-mortem levels of DOPAC were significantly reduced while DOPEG remained almost normal. An attempt was made to selectively destroy the LC cell bodies by a unilateral injection of ibotenic acid (10 micrograms). Eight to 15 days after injection, no current was detectable in the injected side although it was still present in the contralateral intact side. Post-mortem levels of DOPAC and DOPEG levels of the lesioned side were 29% and 17%, respectively, of those in the intact side. Thus, we assumed that the in vivo catechol current in the LC comes from the oxidation of DOPAC most probably synthesized by the noradrenergic cell bodies.

3,4-Dihydroxyphenylacetic Acid↗

Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. II. Pharmacological and behavioral study.

Differential pulse voltammetry used in combination with electrochemically treated carbon fiber electrodes allowed us to detect catechols in the locus coeruleus (LC) of conscious freely moving rats. A micromanipulator cemented on the rat skull was designed in order to implant carbon fiber electrodes without anesthesia. Voltammograms were recorded every 2 min for 5 h. After the in vivo experiments electrodes were tested in 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylglycol (DOPEG) and noradrenaline (NA) solutions. The catechol peak recorded from LC was suppressed by pargyline treatment and slightly reappeared after inhibition of the NA reuptake by desipramine (DMI). This reappearing signal was attributed to NA and estimated at a concentration 50 nM NA. Various drug treatments (piperoxane, haloperidol, clonidine, DMI and reserpine) allowed us to further support the conclusion of part I of this study: the catechol peak recorded from LC is mainly due to DOPAC synthesized by LC noradrenergic neurons. This DOPAC signal corresponded to a DOPAC concentration which reached 23 microM when the whole active part of the electrode was implanted in the LC. In addition to this pharmacological study, data from stress experiments pointed out a striking parallel between the variations of the DOPAC signal and those of the activity of LC noradrenergic neurons as revealed by reported electrophysiological studies.

3,4-Dihydroxyphenylacetic Acid↗

Origin and influence of the serotoninergic innervation of the subcommissural organ in the rat.

The origin of the serotoninergic innervation of the rat subcommissural organ was studied using radioautography of tritiated serotonin and biochemical determination of endogenous serotonin content after electrolytic lesions of raphe nuclei. The results suggest that this innervation is mainly derived from nuclei raphe centralis superior and raphe dorsalis, each nucleus contributing about one-third of the input. A possible contribution from nucleus raphe pontis is also suggested. Given the different patterns of innervation revealed by silver staining of nerve fibers and the different patterns of secretory activity observed with histochemical methods after the electrolytic lesions, the following working hypothesis is formulated. Nucleus raphe dorsalis would inhibit the synthesis of secretory material in the rat subcommissural organ via medium-sized serotoninergic fibers restricted to the hypendymal region, whereas nucleus raphe centralis superior might inhibit the release of secretory material via rather thin serotoninergic fibers reaching the nuclear level of the ependyma. This hypothesis is in line with the inhibitory effect postulated for the serotoninergic innervation in the rat subcommissural organ in early investigations using serotonin neurotoxins.

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Localization of substance P- and enkephalin-like immunoreactivity in relation to catecholamine-containing cell bodies in the cat dorsolateral pontine tegmentum: an immunofluorescence study.

The distribution of tyrosine hydroxylase-, substance P- and enkephalin-immunoreactive neurons in the cat dorsolateral pons was studied using the indirect immunofluorescence method of Coons. To allow for the visualization of substance P- and enkephalin-immunoreactive cell bodies, colchicine was injected either in the ventricular space or in the cerebral tissue. The distribution of the tyrosine hydroxylase-immunoreactive cell bodies corresponded with the well-known distribution of catecholamine cells in this area of the brain. The observation of adjacent sections treated separately with tyrosine hydroxylase- and enkephalin-antiserum revealed that most catecholaminergic cells contain enkephalin-immunoreactivity. In addition to this catecholamine-enkephalin cell population, a moderate number of substance P-immunoreactive cell bodies was found in dorsolateral pons. The peribrachial nuclei were found to be densely supplied with substance P- and enkephalin-immunoreactive fibers, whereas the medial subdivisions, which contain the majority of the catecholamine cells in the dorsolateral pons, display a moderate number of immunoreactive fibers. These results are suggestive of interactions between peptide-containing and catecholaminergic neurons and also between-peptide-containing and non-catecholamine-containing neurons in the cat dorsolateral pons.

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Light- and electron-microscopic immunocytochemistry of glutamic acid decarboxylase (GAD) in the basal hypothalamus: morphological evidence for neuroendocrine gamma-aminobutyrate (GABA).

GABAergic cells and axon terminals were localized in the basal hypothalamus of differnet species (rat, mouse and cat), by means of an immunocytochemical approach using a specific and well-characterized antiserum to the GABA biosynthetic enzyme, glutamate decarboxylase. Light-microscopic visualization was performed with an indirect immunofluorescence method and electron-microscopic observations were made on material with pre-embedding staining and use of the peroxidase-antiperoxidase procedure. At the light-microscopic level, a dense immunofluorescent plexus was observed over both the medial and lateral parts of the external layer of the median eminence. The labelling extended from the rostal part of the median eminence up to the pituitary stalk. Over the subependymal and internal layers only a few immunoreactive dots were visible, except around the blood vessels where they appeared more concentrated. Immunoreactive varicosities could be found following the outlines of the capillary loops and lining tanycyte processes, especially in the median eminance midportion. At the electron-microscopic level, the immunolabelling was exclusively found over neuronal profiles in the median eminence. The latter represented a small fraction of the total number of varicosities visible on the same section. Labelled profiles typically contained numerous small clear synaptic vesicles and only a few or no dense-core vesicles. In the subependymal and internal layers, rare labelled endings were found close to ependymal cells or among transversally cut fibers, respectively. In the palisadic zone, elongated positive boutons were visible intermingled with bundles of unlabelled axons and glial or ependymal processes. In the neurohemal contact zone, immunoreactive endings were observed among unlabelled neurosecretory endings in close vicinity to fenestrated capillary perivascular space. Small moderately intense immunofluorescent varicosities were observed all over the hypothalamus. The density of the glutamate decarboxylase-positive network was higher than in most diencephalic regions. Intraventricular or topical injection of colchicine allowed the visualization of small lightly immunoreactive cells in the diffusion area of colchicine. In the arcuate nucleus labelled axonal endings containing small pleomorphic synaptic vesicles and sometimes a few dense-core vesicles were observed at the electron-microscopic level. Typical synaptic junctions were commonly found between positive endings and unlabelled perikarya, or more frequently, unlabelled dendrites. These findings show that glutamate decarboxylase-containing endings are localized ed in several strategic sites for potential GABAergic neuroendocrine regulations. The GABAergic endings found among neurosecretory endings in the neurohemal contact zone may provide the morphological support for the release of gamma-aminobutyrate into the portal blood flow as an hypothalamic hypophysiotropic hormone.

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Evidence for the presence of enkephalin in catecholaminergic neurones of cat locus coeruleus.

The distribution of enkephalin (Enk) and tyrosine hydroxylase (TH) immunoreactivity in the cat locus coeruleus (LC) has been studied with indirect immunofluorescence technique. After intratissular injection of colchicine numerous enkephalin-containing cell bodies were seen throughout the rostrocaudal extent of the LC complex. Comparison of 8 micrometers-thick consecutive sections treated with antiserum to Enk of TH, a specific marker for catecholaminergic neurons, shows that most cells containing TH also present Enk immunoreactivity. This study provides the first evidence for the coexistence of enkephalin and catecholamine in CNS neurons.

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A new double labeling technique using tyrosine hydroxylase and dopamine-beta-hydroxylase immunohistochemistry: evidence for dopaminergic cells lying in the pons of the beef brain.

Despite numerous data on the organization of the central catecholaminergic (CA) system, the nature of many CA terminal fields, fiber pathways and cell bodies, remains unknown. The discrepancies between the results obtained by CA histofluorescence and dopamine-beta-hydroxylase (DBH) immunofluorescence method suggest that certain areas described as noradrenergic (NA) may be in fact dopaminergic (DA). A method to solve this problem is to compare the localization of the two CA synthetizing enzymes: tyrosine hydroxylase (TH) and DBH. The first enzyme is a marker of all CA neurons, whereas the second one is a specific marker of noradrenergic and adrenergic neurons. We decided to develop a technique which allows visualization of both antigens on a same section. We chose to combine immunofluorescence and immunoperoxidase techniques which yield very different staining. The optimal conditions for this double labeling technique were developed in bovine brain, the homologous system for our antibodies. The results obtained by this procedure provide evidence that DA cells together with NA cells extend throughout the rostral part of the pons, and outline a successful technique for a more extensive investigation of the DA system. The same method should be applicable to other investigations involving other antigens.

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Alterations in tyrosine hydroxylase activity elicited by raphe nuclei lesions in the rat locus coeruleus: evidence for the involvement of serotonin afferents.

The time course of the variations in tyrosine hydroxylase (TH) activity was measured in the rat locus coeruleus (LC) after lesions of the nucleus raphe dorsalis (NRD), nucleus raphe centralis superior (NRCS) and nucleus raphe pontis (NRP). A certain number of lesions were performed in the raphe magnus (RM), the caudal and rostral NRP and the caudal and rostral NRCS, lateral to raphe nuclei and in adrenalectomized animals. The serotonin (5-HT) content in the LC was also determined after these lesions. Only raphe nuclei producing significant decreases in the 5-HT content in the LC are successful in provoking increases in the TH activity in the LC, thus these results suggest that the noradrenaline (NA) synthesis in the LC may be regulated by 5-HT afferents. Moreover, intraventricular injections of 5,6-dihydroxytryptamine (5,6-DHT) and administration of parachlorophenylalanine (PCPA) also produce significant increases in TH in the LC. After immunotitrations of TH in the LC it was shown that, with exception of a high dose of 5,6-DHT (75 micrograms), all these treatments provoke an increase in the concentration of the enzyme. It therefore seems that one of the functional roles of 5-HT in the LC could be the regulation of the concentration of TH.

5,6-Dihydroxytryptamine↗

Immunocytochemical evidence for the existence of GABAergic neurons in the nucleus raphe dorsalis. Possible existence of neurons containing serotonin and GABA.

It has been established that nerve cell bodies of the nucleus raphe dorsalis (NRD) belong to ascending 5-hydroxytryptamine systems. These neurons could be modulated by GABAergic interneurons or interposed GABA neurons. A high glutamate decarboxylase (GAD) activity in the NRD and a specific high-affinity uptake mechanism for GABA suggest the presence of GABA synthesizing elements in the NRD. Anti-GAD antibodies were used by an immunocytochemical procedure to demonstrate the presence of GABAergic elements. Anti-GAD antibodies were previously tested in the cerebellum and substantia nigra. Large amounts of GAD-positive reaction product were observed in the cytoplasm of some neurons (fusiform, ovoid or multipolar) or appeared as punctate deposits apposed to dendrites, soma and dispersed in the neuropil of the NRD. At the electron microscopic level, GAD-positive reaction product was observed within the cytoplasm of numerous somata in sections from colchicine-treated rats. GAD-positive staining was observed in numerous fibers or axonal terminals and two types of morphologically different fibers could be distinguished. The first displays small clear vesicles and few large granular vesicles (LGV) (80-100 nm), the second displays only clear round vesicles (40-60 nm). After 5,7-dihydroxytryptamine treatment (a neurotoxic for 5-HT terminals), the immunocytochemical labeling is much decreased. Some reactive neurons are still dispersed in the nucleus but the fibers containing LGV are no longer observed. These results strongly suggest that some neuronal elements in the NRD are morphologically, pharmacologically and anatomically similar to 5-HT neurons described at this level. Such cell elements could possess a double GABA and 5-HT potentiality. If this is not the case, a population of GABA neurons could be sensitive to 5,7-DHT and so have the capacity to take up 5-HT. The other reactive elements, insensitive to 5,7-DHT, could represent the GABAergic interneurons postulated at this level. Numerous GAD positive fibers or axon terminals were observed in synaptic contact with dendrites, axons or soma of other neurons. The chemical nature of the neuronal postsynaptic elements remains unknown. These findings strongly support the hypothesis for GABA-mediated inhibition in the NRD.

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Identification of cDNA clones coding for rat tyrosine hydroxylase antigen.

Five recombinant DNA plasmids have been constructed that contain structural gene sequences for rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]. Rat pheochromocytoma PC 12 cell line, which contains relatively high levels of catecholamine-synthesizing enzymes, was used to purify RNA. TyrOHase cDNA clones were identified by screening 350 cDNA clones constructed from partially purified TyrOHase mRNA. A rapid and powerful screening of the recombinant clones by differential colony hybridization was possible because TyrOHase is a tissue-specific protein. The final selection relied on the ability of cDNA inserts to hybridize specifically to TyrOHase mRNA as judged by cell-free translation and immunoprecipitation. Blot hybridization analysis of polyadenylylated RNA from PC 12 cells indicated a major mRNA species of 1.9 kilobases. A species of the same size was identified from a human pheochromocytoma tumor, indicating a crossreactivity between rat TyrOHase cDNA and human TyrOHase mRNA.

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Differential pulse voltammetry in brain tissue. I. Detection of 5-hydroxyindoles in the rat striatum.

In vitro, differential pulse voltammetry combined with electrochemically treated carbon fiber electrodes enabled detection, in different solution of 5-hydroxyindole compounds, of an oxidation peak 3 at +300 mV. In vivo, a striatal peak 3 was also recorded at this potential. Electrolytic or 5,7-dihydroxytryptamine lesions interrupting the medial forebrain bundle (MFB) were followed by a decrease of 65% and 64% in peak height, but not elimination of the peak. Biochemical determinations were significantly correlated to the peak 3 measurements. The existence of peak 3 as well as hydroxyindole compounds in blood suggested a blood contamination under the experimental conditions employed. This possibility is confirmed both by the complete disappearance of striatal peak 3 in animals with the MFB lesioned and surgically prepared a week before recordings, and by biochemical measurements in parachlorophenylalanine-treated or perfused (phosphate-buffered saline solution) animals.

3,4-Dihydroxyphenylacetic Acid↗

Differential pulse voltammetry in brain tissue. II. Detection of 5-hydroxyindoleacetic acid in the rat striatum.

Differential pulse voltammetry with electrochemically treated carbon fiber electrodes was used to study, in anesthetized rats, the alterations of the striatal electrochemical signal appearing at +300 mV (peak 3), following pharmacological manipulation of the animals. Decreases in peak 3 were obtained after injections of NSD 1015, Ro 4-4602 and Clorgyline. This, in conjunction with biochemical measurements, suggested that 5-hydroxyindoleacetic acid (5-HIAA) is, to a considerable extent, responsible for this electrochemical signal. Increases in peak 3 obtained after reserpine, L-tryptophan and D,L-5-HTP, as well as biochemical data reinforced this hypothesis, as did the detection of peak 3 in cerebrospinal fluid and its increase after probenecid injection. The occurrence of a feed-back mechanism is discussed following injection of D,L-5-HTP.

5-Hydroxytryptophan↗

Voltammetry in the striatum of chronic freely moving rats: detection of catechols and ascorbic acid.

Differential pulse voltammetry used in combination with an electrochemically treated carbon fiber electrode allowed to detect ascorbic acid (AA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum of unanaesthetized freely moving rats. Carbon fiber electrodes were implanted under light halothane anaesthesia. When the rat recovered from anaesthesia, voltammograms were recorded every 2.5 min for about 5 h. During the last 3 h the signals corresponding to AA and DOPAC appeared stable and reproducible, allowing comparisons between control and pharmacological experiments. After the in vivo experiments, the electrodes were calibrated in AA and DOPAC solutions. Striatal AA and DOPAC levels were thus estimated to be 306 and 17.7 microM respectively. DOPAC signals were increased by haloperidol and chloral hydrate, decreased by amphetamine and suppressed by pargyline. In pargyline pretreated rats the catechol signal slightly re-appeared after amphetamine injections. This signal was attributed to dopamine (DA) and estimated to a concentration less than 50 nM DA. The AA current recorded in the striatum was not modified when dopaminergic terminals selectively degenerated. The AA signal was decreased by chloral hydrate or halothane anaesthesia and increased by amphetamine injections. This latter effect depended on the presence of dopaminergic terminals. This shows that the increasing current effect of amphetamine, previously observed by non-selective techniques, should be attributed to AA rather than DA. These results confirm that the catechol compound electrochemically detected in the striatum is the direct metabolite of the DA, i.e. DOPAC, and support the thesis of a functional relationship between AA levels and dopaminergic neurotransmission.

3,4-Dihydroxyphenylacetic Acid↗

In vivo electrochemical detection of catechols in several dopaminergic brain regions of anaesthetized rats.

Differential pulse voltammetry (DPV) was used in conjunction with electrochemically treated carbon fibre microelectrodes to monitor in vivo the catechols (CA) levels in the substantia nigra (SN), ventral mesencephalic tegmentum (VMT) and nucleus accumbens of anaesthetized rats. The current corresponding to CA oxidation was always closely related to the anatomical localisation of the dopaminergic neurons e.g. it was maximal in tht pars compacta of the SN. In addition, the current was selectively suppressed in all regions after 6-OHDA-induced degeneration of the dopaminergic neurons and after pargyline treatment. These findings together with our previous results suggest that 3,4-dihydroxyphenylacetic acid (DOPAC) is the main contributor to the CA oxidation current measured in these areas. Haloperidol induced a significant increase of the CA signal in the nucleus accumbens (+70%) but not in the SN or VMT. The DOPAC concentrations declined exponentially after pargyline administration. The fractional rate constant of DOPAC, computed from these results appears to be similar in all regions studied except for n. accumbens where the low rate of disappearance might be responsible for the high DOPAC concentration measured in this area. In conclusion, DPV with electrochemically treated carbon fibre electrodes is a useful means of monitoring extracellular DOPAC levels in dopaminergic brain regions.

3,4-Dihydroxyphenylacetic Acid↗

The viability of transplants of mesencephalic raphe nuclei in the IVth ventricle of the adult rat.

Mesencephalic raphe nuclei were transplanted via the cisterna magna near the locus coeruleus. The transplants were observed at different time intervals ranging from 24 h to 4 months. Microscopic observations revealed that from 3 weeks to 4 months the transplant developed with a well-defined neural organization. Radioautographic results showed that some of the neurons in the implant at 3 weeks captured and retained [3H] serotonin. These results indicate that the intact IVth ventricle seems to be a likely culturing floor for mesencephalic raphe nuclei.

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[In vitro synthesis of tyrosine hydroxylase].

RNA from PC 12 cells was translated in a cell-free system and the peptides synthesized in vitro were analyzed by immunoprecipitation with an anti-T.H. serum raised in Rabbit. Analysis of the immunoprecipitate on a SDS-polyacrylamide gel reveals a major band with a molecular weight of 62000 dalton. A protein of the same molecular weight was also precipitated from a PC12 cell lysate. This molecular weight of 62000 dalton corresponds to the reported for tyrosine hydroxylase purified from PC12 cells.

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