A radioautographic and immunocytochemical study of the GABA systems of the habenula complex in the rat.

Radioautography of [(3)H]GABA accumulation and immunocytochemistry of glutamate decarboxylase have been used to study anatomically and morphologically the GABA system of the rat habenular (Hb) complex. Radioautographic visualisation of GABA specific neurons show a very high innervation of the complex including both stria medullaris (SM), the habenular commissure and the periventricular thalamic fibers (FPVT). A massive labeled fiber system in the SM appears to divide into two branches when it reaches the Hb nuclei: a part of fibers continue their course dorsally to the nuclei up to the habenular commissure; other fibers enter the Hb lateralis or run along the ventral Hb medialis at the level of FPVT. The staining is markedly diminished in the entire complex in response to SM lesions. In the Hb lateralis, the radioautographic-positive reaction is mainly bound to labeled fibers or axonal varicosities. However GAD immunocytochemistry reveals some GAD-positive cell bodies in the ventro-median portion of the nucleus. In the Hb medialis the radioautographic and immunocytochemical staining is observed in the neuropile between the unlabeled large cell bodies. In the subependymal layer bundles of processes are strongly labeled and form a continual strain behind the unlabeled ependymocytes. Three types of reactive terminals have been differentiated based on size and shape of vesicles. Some of them are exclusively characterized by clear round vesicles and probably have their origin in the septum. Others contain clear vesicles and some large dense-cored vesicles and disappear after mesencephalic Raphe lesions or 5,7-dihydroxytryptamine treatment. They could correspond to terminals of raphe neurons with a double potentiality GABA and 5HT. The last exhibit mainly a dense population of large dark-cored granules similar to the ones found in neurosecretory nerve endings. However numerous fibers morphologically similar to the reactive fibers are unlabeled.

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.

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.

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.

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.

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.

Evidence for cysteine sulfinate as a neurotransmitter.

The Na+-independent binding of L-[3H]cysteine sulfinate and L-[3H]cysteine sulfinate uptake were investigated in rat brain membranes and vesicles. Specific binding of L-[3H]cysteine sulfinate was saturable and occurred by a single high affinity process with a Kb of 100 nM +/- 9 and a capacity (Bmax) of 2.4 +/- 0.22 pmol/mg protein. Sodium ions were found to have a biphasic effect; low concentrations (in the range of 0.1-3 mM) induced a marked inhibition of the binding whereas higher concentrations (10-300 mM) resulted in a dose-dependent stimulation of binding. The inhibition potency, expressed as the Ki values of a wide range of compounds with known pharmacological activities was tested. L-Cysteine sulfinate was the most potent inhibitor being 3-fold more potent than L-glutamate and 80 times more potent than L-aspartate. The regional distribution of the binding of L-[3H]cysteine sulfinate in the brain was found to be heterogeneous. These results provide the first evidence for an interaction of cysteine sulfinate with specific receptor sites on the synaptic membrane. The rate of L-[3H]cysteine sulfinate uptake shows a biphasic dependence on the concentration of L-cysteine sulfinate, corresponding to a high affinity (27.2 microM) and a low affinity (398 microM) transport system. The maximum L-[3H]cysteine sulfinate uptake is reached at 2 min. The reversibility of this transport was demonstrated. The L-[3H]cysteine sulfinate uptake increases as a function of the sodium concentration. Chloride and potassium ions stimulate the uptake. The decrease or increase in the electrical membrane potential (delta psi) caused by replacing the chloride ions by the sulfate or sulfocyanate ions respectively leads to a decrease or increase in the rate of uptake. Increase in the extravesicular osmolarity leads to a decrease in the extent of L-[3H]cysteine sulfinate accumulation. Amino acids with an acidic group in position omega were found to be potent inhibitors (the most potent being L-aspartate). The length of the carbon chain also has a bearing on the inhibitory effect. The regional distribution of L-[3H]cysteine sulfinate uptake in the brain was heterogeneous. These results demonstrate the existence of a high affinity system which may correspond to the transmitter inactivation. Binding and uptake sites are distinguishable as evidenced by the affinity constants, the ionic and pharmacological effects and the different regional distributions in the brain. Finally, these results give further evidence for a neurotransmitter role of L-cysteine sulfinate.

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.

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.

Tubulin dipole moment, dielectric constant and quantum behavior: computer simulations, experimental results and suggestions.

We used computer simulation to calculate the electric dipole moments of the alpha- and beta-tubulin monomers and dimer and found those to be |p(alpha)| = 552D, |p(beta)| = 1193D and |p(alphabeta)| = 1740D, respectively. Independent surface plasmon resonance (SPR) and refractometry measurements of the high-frequency dielectric constant and polarizability strongly corroborated our previous SPR-derived results, giving Deltan/Deltac approximately 1.800 x 10(-3)ml/mg. The refractive index of tubulin was measured to be n(tub) approximately 2.90 and the high-frequency tubulin dielectric constant k(tub) approximately 8.41, while the high-frequency polarizability was found to be alpha(tub) approximately 2.1 x 10(-33)C m(2)/V. Methods for the experimental determination of the low-frequency p are explored, as well as ways to test the often conjectured quantum coherence and entanglement properties of tubulin. Biobits, bioqubits and other applications to bioelectronics are discussed.

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.