Immunocytochemical evidence for the presence of enzymes synthesizing GABA and GHB in the same neuron.

A specific and sensitive immunocytochemical double staining for visualization of glutamate decarboxylase (GAD) and semialdehyde succinate reductase (SSR(2)) in the same brain section has been developed. SSR(2) is the enzyme responsible for the transformation of succinic semialdehyde into ?-hydroxybutyrate (GHB). GAD was detected using specific rabbit GAD-antibodies and unlabeled antibody enzyme peroxidase antiperoxidase, and SSR(2) using specific guinea-pig SSR(2) antibodies conjugate to a fluorescein-labeled second antibody. The coexistence of GAD and SSR(2) in the same neuron was demonstrated by a peroxidase reaction superimposed on fluorescent compounds. Cell bodies containing both antigens were observed in the cerebellum, dorso-median hypothalamus and raphe nuclei. GHB is present in most GABA containing neurons. Some neurons contain only SSR(2); these neurons may synthesize GHB by an active uptake of GABA.

Evidence for the localization of 5HT1A binding sites on serotonin containing neurons in the raphe dorsalis and raphe centralis nuclei of the rat brain.

Precise anatomical distribution of 5HT1 binding sites has been investigated in the nuclei raphe dorsalis, raphe centralis and locus caeruleus of the rat brain. An original pattern of distribution was observed in the raphe nuclei, closely correlated to the already known distribution of 5HT containing elements. This pattern, more pronounced when 5HT1A sites were labelled, completely disappeared after lesioning by 5, 7DHT indicating the presence of this subtype of 5HT1 binding sites on 5HT containing neurons. It is postulated that these 5HT1A sites correspond in these raphe nuclei to 5HT autoreceptors.

Immunocytochemical localization in rat brain of the enzyme that synthesizes ?-hydroxybutyric acid.

?-Hydroxybutyric acid, a reductive catabolite of GABA, has numerous neuropharmacological and neurophysiological properties when injected systematically to animals. Recently, a specific succinic semialdehyde reductase (SSR2) has been isolated from rat brain. This enzyme specifically produces [(3)H]?-hydroxybutyrate from [(3)H]GABA when incubated in vitro with rat brain tissue slices. A specific antibody against this enzyme has been raised in the rabbit and employed to localize by immunocytochemical procedures the sites of ?-hydroxybutyrate synthesis in two regions of rat brain, the nucleus Raphe dorsalis and the median hypothalamus. Light microscopy reveals the presence of numerous SSR2-positive reactions in the cytoplasm of fusiform or ovoid cells. High magnification shows that only neurons of varous sizes are stained; the cytoplasm is uniformly labelled with a few punctate deposits. At the electron microscopic level, some staining appears in the somata of neurons and in fibres or axonal terminals.

Immunohistochemical evidence for neuronal and non-neuronal synthesis of GABA in the rat subcommissural organ.

In the past few years, several studies have demonstrated in the rat subcommissural organ the presence of nerve endings and modified ependymocytes showing an uptake of [(3)H]GABA. The present work was performed to demonstrate in this cerebral zone the possibility of a GABA synthesis by the immunohistochemical localization of glutamate decarboxylase (GAD). GAD-positive reaction was detected with unlabelled antibody-enzyme peroxidase anti-peroxidase. Some nerve terminals containing either clear round vesicles, or sometimes clear round vesicles and some large granular vesicles, exhibited a positive staining. These terminals could belong to GABAergic inputs in the subcommissural organ. The few reactive terminals containing some granular vesicles could be related to the serotoninergic input as suggested previously (Gamrani et al., 1981). Several ependymocytes of this structure contained GAD-like positive reaction; these cells are also capable of taking up [(3)H]GABA (Gamrani et al., 1981) and present neuronal properties with regard to GABA. However, the presence in their cytoplasm of ?? enolase, a specific glial marker, related them to glial elements. The presence of GABA in these ependymocytes suggests a modulating function of GABA on the secretory activity of the subcommissural organ.

Benzodiazepine binding sites: localization and characterization in the limbic system of the rat brain.

The distribution of benzodiazepine binding sites was analysed in limbic structures of rat brain by quantitative radioautography of brain sections incubated with 3H-flunitrazepam (3H-FLU). Quantitative estimation of the binding parameters was made in each range of postero-anterior sections taken. Distribution of 3H-FLU binding sites was found to be rather homogeneous in most of the structures examined but there were regional differences which resulted from variations in the densities of sites rather than in their affinities. A particular distribution pattern of 3H-FLU binding sites was observed in the cingulate cortex contrasting with the homogeneous postero-anterior distribution measured in other cortical areas in the same slices. A significantly greater density of sites was found in the anterior part of the structure as compared to the posterior part. This difference, which corresponds to a change in the density of sites without alteration of their apparent affinity and occurs at a precise anatomical level, is discussed with reference to the anatomical organization of this brain structure and to its possible functional implications.

Depolarization-evoked release of gamma-hydroxybutyrate from rat brain slices.

The release of gamma-hydroxybutyrate from preloaded rat brain striatal slices was investigated. K+-induced depolarization caused an efflux of gamma-hydroxybutyrate of about 50 fmol min-1 mg-1 (wet weight), but in a Ca2+-free medium containing Mg2+, the evoked release was reduced by 50-60%. The release was higher when 100 microM veratridine was used as a depolarizing agent. The efflux of gamma-hydroxybutyrate is related to veratridine and K+ concentration, and is strongly inhibited by 10 microM tetrodotoxin. The Ca2+ channel blocker verapamil induces a large decrease in the efflux of gamma-hydroxybutyrate after both K+- and veratridine-induced depolarization. These results are in favour of a possible transmitter function for gamma-hydroxybutyrate in rat striatum.