J.F. Pujol

Immunohistochemical evidence for the presence of γ-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.

GABA-accumulating neurons in the nucleus raphe dorsalis and periaqueductal gray in the rat: A biochemical and radioautographic study

The possibility of a GABAergic innervation of the nucleus raphe dorsalis (NRD) has been investigated by using the following approaches: (i) the identification of the principal neuronal groups afferent to the NDR by using horseradish peroxidase retrograde transport, (ii) the determination of glutamate decarboxylase activity (GAD) in the NRD after lesioning these groups or their putative pathways, and (iii) the radioautographic identification of terminals axons and nerve cells accumulating intraventricularly injected [3H]GABA. The hypothesis of a local GABAergic network is supported by the failure to obtain important changes in GAD after lesions of NRD afferents and the presence in this nucleus of terminals, fibers and nerve cell bodies accumulating [3H]GABA. It appears that these GABA-accumulating neurons could represent a portion of aperiventricular GABAergic system in the periaqueductal gray and the pontine ventricular gray.

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

Abstract GABAergic cells and axon terminals were localized in the basal hypothalamus of different 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. Lightmicroscopic 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 rostral 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 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 γ -aminobutyrate into the portal blood flow as an hypothalamic hypophysiotropic hormone. Alternatively, neurosecretory cells might be under GABAergic control expressed either at their terminal level within the median eminence or the cell body level within the parvicellular hypothalamic nuclei.

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.

The efferent connections of the nucleus raphe centralis superior in the rat as revealed by radioautography

By chronically implanting a glass micropipette filled with tritiated leucine in the raphe centralis superior of the rat, the projection of this nucleus was traced by radioautography. The majority of the ascending projections were located within the ventral tegmental area and, further rostrally, the median forebrain bundle. Along the course of this bundle numerous fibers branched successively into the mammillary peduncle, the fasciculus retroflexus, the stria medullaris, the fornix and the cingulum. The most significant projections included the ones to the interpeduncular nucleus, the mammillary bodies, the habenular nuclei and the hippocampus. No projections were detected in the striatum, the cortex piriformis or the amygdala. Descending projections diffused to the pontine reticular formation and central gray through the medial and the dorsal longitudinal bundles. In addition widespread projections were also seen in nuclei located near the raphe centralis superior: raphe nuclei, dorsal and ventral tegmental nuclei.

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 +0.1v

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.

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.

Differential pulse voltammetry in brain tissue. II. Detection of 5-hydroxyindolacetic 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.

Localization of substance P- and enkephalin-like 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.

Neuron-specific enolase as a marker of neuronal lesions during various comas in man ☆

The detection of neuron-specific enolase in biological fluids has been investigated as an indirect marker of neuronal damage in man. This protein was measured by a sandwich enzymoimmunoassay in serums and cerebrospinal fluids from patients with consciousness disorders of various aetiologies. Neuron-specific enolase level was significantly increased in sera from patients with comas resulting from anoxemia, head injury, septic state, cirrhosis and fulminant hepatitis. On the other hand, patients with meningitis (affection not normally accompanied with neuronal lesion) exhibited no change of this marker level. The statistical analysis of our results suggests that, in neurological disorders, the neuron-specific enolase levels in cerebrospinal fluid could have some prognostic value. The correlation between its level in cerebrospinal fluid and in serum was also demonstrated. Neuron-specific enolase increase in biological fluids thus represents a useful and promising marker to biochemically characterize various strokes possibly resulting in neuronal damage.