Selective tonicity-induced expression of the neutral amino-acid transporter SNAT2 in oligodendrocytes in rat brain following systemic hypertonicity.

Sodium-coupled neutral amino-acid transporter member 2 (SNAT2) belongs to the family of neutral amino-acid transporters. SNAT2 is encoded by the gene Slc38a2, whose expression was reported to increase in vitro in fibroblasts, endothelial and renal cells exposed to a hypertonic medium. SNAT2 tonicity-induced expression brings about cellular accumulation of amino-acid, which contributes to osmoadaptation to hypertonicity. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic imbalances in blood plasma, we have investigated, through immunocytochemistry, SNAT2 expression in brain of rats subjected to systemic hypertonicity. Following prolonged systemic hypertonicity (24 h), small, strongly immunolabeled elements were observed that were not present in sham-treated animals. They were evenly distributed in the gray matter, with a lower density in the forebrain and a higher density in the brain stem. However the highest density by far was observed in white matter, where they were frequently aligned in chain-like rows. These observations suggested an oligodendrocyte location that was further established by double immunofluorescent labeling, using the oligodendrocyte phenotypic markers 2'-3'-cyclic nucleotide 3'phosphodiesterase and carbonic anhydrase II. SNAT2-positive elements were found associated with oligodendrocyte cell bodies, while oligodendrocyte processes were devoid of labeling. A quantitative analysis performed in the cerebral cortex indicated that virtually all SNAT2-positive elements were associated with oligodendrocyte cell bodies and conversely that the overwhelming majority of oligodendrocytes showed SNAT2 immunolabeling. The tonicity-induced expression of SNAT2 was not observed following acute systemic hypertonicity (6 h). Our results suggest that the osmoadaptation of brain oligodendrocytes to hypertonicity relies upon amino-acid accumulation through the tonicity-induced expression of SNAT2. The possible significance of these findings in relationship to the selective loss of oligodendrocytes observed in osmotic demyelination syndrome is discussed.

Large discrepancies in cellular distribution of the tonicity-induced expression of osmoprotective genes and their regulatory transcription factor TonEBP in rat brain.

Osmoprotective genes are tonicity-activated genes involved in cellular osmoadaptation to hypertonicity and considered to be regulated by a specific transcription factor called tonicity-responsive enhancer-binding protein (TonEBP). In the brain we had previously established that TonEBP was expressed and tonicity-induced in neurons only. Here we have compared in various brain regions of rats subjected to systemic hypertonicity, the cellular expression of TonEBP through immunocytochemistry and the cellular expression of osmoprotective genes, namely aldose reductase (AR), sodium-dependent myo-inositol transporter (SMIT), betaine/GABA transporter (BGT1) and taurine transporter (TauT), by in situ hybridization using non-radioactive digoxigenin-labeled riboprobes. In neurons where TonEBP was strongly tonicity-induced, AR-mRNA labeling was strongly increased in some subsets (e.g. hippocampus pyramidal cells, cerebellar Purkinje cells and neurons of the hypothalamic magnocellular nuclei) but remained undetectable in some other subsets (e.g. neurons in cerebral cortex). Tonicity-induced AR-mRNA labeling was observed only several hours after the tonicity-induced expression of TonEBP. SMIT-mRNA labeling was tonicity-induced as densely and evenly distributed dots in neuron poor regions (e.g. cerebral cortex layer I and hippocampus stratum lacunosum-moleculare). The tonicity-induced expression of SMIT-mRNA may thus occur in non-neuronal cells, presumably astrocytes, where TonEBP is neither significantly expressed, nor tonicity-induced. In neurons showing a strong tonicity-induced expression of TonEBP, no SMIT-mRNA labeling was observed. BGT1-mRNA and TauT-mRNA labeling could not be detected, even after systemic hypertonicity. The present work reveals large discrepancies between the cellular distribution of the tonicity-induced expression of osmoprotective genes and that of their regulatory transactivator TonEBP. Depending on the cell subsets and the osmoprotective genes, TonEBP may appear insufficient or conversely unnecessary for the tonicity-induced activation of an osmoprotective gene. Altogether our results show that brain cells, even from the same class, activate distinct osmoprotective genes through distinct activation processes to adapt to hypertonicity.

Differential cellular distribution of tonicity-induced expression of transcription factor TonEBP in the rat brain following prolonged systemic hypertonicity.

In a previous work performed on cerebral cortex and hippocampus we reported that tonicity-responsive enhancer binding protein (TonEBP), originally identified as a transactivator of osmoprotective genes involved in osmoadaptation of renal cells, was induced in neurons only, but to varying levels, following acute systemic hypertonicity. Whether or not this cellular specificity reflected a unique ability of neurons or a differential time course among brain cells for tonicity-induction of TonEBP was investigated throughout the brain in this study by subjecting the animals to prolonged systemic hypertonicity. In normal rats, TonEBP immunolabeling and TonEBP-mRNA in situ hybridization labeling showed a widespread, uneven and parallel distribution. TonEBP was expressed primarily in the cell nuclei of neurons, where it was heterogeneously distributed in a nucleoplasmic and a granular pool. In rats subjected to prolonged systemic hypertonicity, TonEBP labeling increased in the cell nuclei of neurons only. The tonicity-induced expression of TonEBP for a given cell group of neurons was rather uniform but varied greatly among neuronal cell groups and was positively correlated with the average size of the cell nuclei, as determined by quantitative analysis of digitized images. The detailed distribution of tonicity-induced expression of TonEBP is reported throughout the brain. In normal rats, a very minor proportion of non-neuronal cells, identified as a subset of astrocytes and possibly oligodendrocytes, showed faint nuclear immunolabeling, which however did not increase in hypertonic animals. Ependymocytes, capillary endothelial cells, and microglial cells showed no TonEBP labeling, even in hypertonic animals. Altogether our data indicate that neurons, albeit possibly to a varying extent, are the only brain cells able to use TonEBP-mediated processes for adaptation to a systemic hyperosmotic unbalance.

Taurine biosynthetic enzymes and taurine transporter: molecular identification and regulations.

Many biological effects of taurine rely upon its cellular concentration, which is primarily controlled by taurine biosynthetic enzymes cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD) and taurine transporter (TauT). The cloning of CDO, CSD and TauT in various species provided first-hand information on these proteins, as well as molecular tools to investigate their regulations. CDO upregulation in hepatocytes in response to high sulfur amino acids appears clearly as the most spectacular among the regulations of the biosynthetic enzymes. Downregulation of TauT activity by activation of PKC appears particularly well documented. A unique serine residue could be identified as a phosphorylation site that leads to an inactive form of TauT. The previously revealed downregulation of TauT expression by taurine and hypertonicity-induced upregulation of TauT expression were shown to result from a modified transcription rate of TauT gene, but the precise molecular mechanisms are not yet formally established. Other regulations of taurine transporter expression were more recently reported, which involve glucose, tumor suppressor protein p53, tumor necrosis factor-alpha, and nitric oxide. This review reports the experimental models and data that support these various regulations but also points out the aspects that remain poorly understood or unknown concerning their molecular basis and physiological significance.

Transcription factor tonicity-responsive enhancer-binding protein (TonEBP) which transactivates osmoprotective genes is expressed and upregulated following acute systemic hypertonicity in neurons in brain.

Tonicity-responsive enhancer-binding protein (TonEBP) was initially identified as a transcription factor involved in adaptation of renal cells to hypertonicity by activation of osmoprotective genes encoding proteins for accumulation of compatible osmolytes. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic disbalances of blood plasma we have investigated through immunocytochemistry TonEBP expression in cerebral cortex and hippocampus of normal rat and rats submitted to an acute systemic hypertonicity or to a prolonged systemic hypotonicity. TonEBP-expressing cells were identified using double immunofluorescence and appropriate cell type markers. Their relative proportion was determined by quantitative image analysis. In normal rats TonEBP expressed primarily in neurons where it was strictly located in the cell nucleus but heterogeneously distributed into a nucleoplasmic pool and a granular pool. In animals made acutely hypertonic TonEBP labeling increased dramatically exclusively in the nuclei of neurons and reached a maximum within 1 h. In hypertonic animals TonEBP labeling covered the whole cell nucleus of virtually all neurons, appeared finely punctuated but was no more granular. Optical density of the labeling as determined by image analysis correlated linearly with the increased plasma osmolality. In animals made hypotonic for several days no conspicuous decrease of TonEBP labeling was observed. In normal animals a very minor proportion of non-neuronal cells showed a faint TonEBP nuclear labeling. This proportion increased slightly in hypertonic animals. Nevertheless these non-neuronal TonEBP-positive nuclei which belonged to oligodendrocytes and to a small subpopulation of astrocytes remained always very weakly labeled when compared with neuron nuclei. Brain capillary endothelial cells as well as microglial cells showed no TonEBP-labeling even in hypertonic animals. Our data demonstrate that in brain TonEBP is significantly expressed and tonicity-overexpressed in neurons and accordingly suggest that neurons only among brain cells accumulate compatible osmolytes through TonEBP-mediated activation of osmoprotective genes to adapt to acute systemic hypertonicity.

Specificity of cysteine sulfinate decarboxylase (CSD) for sulfur-containing amino-acids.

Cysteine sulfinate decarboxylase (CSD) which decarboxylates cysteine sulfinic acid (CSA) to form hypotaurine is thought to be involved in the biosynthesis of taurine. It was recently localized in astrocytes in the cerebellum and hippocampus by immunocytochemistry. Another sulfur-containing amino-acid (SCAA), homocysteic acid (HCA), was also found in astrocytes in these regions. We therefore investigated the specificity of CSD vs CSA and HCA as well as the related analogs homocysteine sulfinic acid (HCSA) and cysteic acid (CA). CSD was immunotrapped from brain and liver tissue supernatant using a specific CSD antiserum and Protein-A Sepharose. It was then incubated with the L-form of the various SCAA. Reaction products were identified and quantified by pre-column o-phthalaldehyde derivatization HPLC. CA and HCA from 2.5 to 25 mM inhibited the formation of hypotaurine from CSA (0.25 mM). Moreover, the inhibition curves were parallel for liver and brain CSD. CA or HCA (25 mM) elicited a near-total inhibition. HCSA did not produce a significant inhibition up to 25 mM. Incubation with 25 mM CSA or CA led to the formation of hypotaurine and taurine, respectively. The ratio of formation of taurine to that of hypotaurine was similar for CSD from liver and brain. In contrast no homotaurine, the decarboxylated reaction product of HCA, could be detected following incubation with 25 mM HCA. According to the sensitivity of the HPLC analysis this indicates that the decarboxylation of HCA, if any, was 130-fold and 50-fold less than that of CSA by CSD from liver and brain, respectively, in our experimental conditions. Similarly, following incubation with HCSA, no new peak appeared on the chromatogram when compared to a blank sample. These results show that CSD from either brain or liver has a high specificity for CSA and CA, which are the SCAA involved in the biosynthesis of taurine. HCA is an inhibitor of CSD but does not appear to be a substrate for CSD in vitro. HCSA is neither a substrate nor an inhibitor of CSD in vitro. Accordingly, CSD is unlikely to play a role in the metabolism of HCA or HCSA in vivo.

Autoantibodies to glutamic acid decarboxylase (GAD) detected by an immuno-trapping enzyme activity assay: relation to insulin-dependent diabetes mellitus and islet cell antibodies.

It has recently been proposed that the islet 64,000 Mr protein autoantigen (64K) of insulin-dependent diabetes mellitus (IDDM) is glutamic acid decarboxylase (GAD). We evaluated, by means of a newly developed immunotrapping enzyme activity assay (ITEAA), the prevalence of circulating GAD-autoantibodies (Ab) in a large population of IDDM patients (n = 168), blood donors (n = 87) and non-diabetic autoimmune patients (n = 40). The latter two groups were used as controls. Overall, GAD-Ab were found in 22% of IDDM patients, but in none of the two control groups (P = 0.007). These specificities were invariably associated with islet cell antibodies (ICA) (31.6% in IDDM with ICA vs 0 in IDDM without ICA, P = 0.0001), and this prevalence was higher in sera with high titer ICA (54.5% in IDDM with ICA greater than 80 JDF-units vs 22.6% of IDDM with ICA 5-80 JDF units; P = 0.002). Moreover, GAD-Ab were associated with the female sex (P = 0.002) and the concomitant presence of thyroid and/or gastric antibodies (P = 0.002). No correlation was observed between GAD-Ab and age of the patients, duration of IDDM, or associated non-organ specific antibodies. Our study indicates that GAD-Ab measured by ITEAA are: (1) detected in a proportion of IDDM patients; (2) strongly associated with ICA; (3) preferentially found in IDDM female patients with autoimmune polyendocrine serology; and (4) detected with lower frequency than that reported for 64K-Ab in IDDM.

Systematic presence of GABA-immunoreactivity in the tubero-infundibular and tubero-hypophyseal dopaminergic axonal systems: an ultrastructural immunogold study on several mammals.

Immunoreactivities for tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA) and, in some cases, glutamic acid decarboxylase (GAD) were detected by light and electron microscopy in axons projecting into the median eminence and pituitary gland of various mammals (rats, mice, guinea pigs, cats, rabbits and hares). Light microscope immunoperoxidase reactions were performed on adjacent semithin sections of plastic-embedded samples. In the median eminence external zone, the distributions of the TH- and GAD- or GABA-immunoreactive endings were very similar in the anterior and lateral areas, while medially the GABA-labelled endings predominated. Comparable distribution patterns were found in the various species examined. In the pituitary gland, the distributions of GABA- and TH-immunoreactivities were superimposable in the intermediate lobes of all species examined, except in the rabbit and hare in which both types of innervation were lacking. For electron microscopy, the immunogold procedure was applied to sections of lowicryl-embedded samples; simultaneous detection of GABA- and TH-immunoreactivities was enabled by recto-verso double labelling with gold particles of distinct diameters. In the median eminence, GABA-immunoreactivity occurred systematically in the TH-positive endings, while distinct GABA-positive/TH-negative axons were also detected. In the intermediate lobe, the colocalization of TH- and GABA-immunoreactivities was a constant feature of the axons innervating the melanotrophic cells in all the species examined, except in the Leporidae. The functional significance of this colocalization remains to be determined.

GABAergic innervation of somatostatin-containing neurosecretory cells of the anterior periventricular hypothalamic area: a light and electron microscopy double immunolabelling study.

Double immunolabelling on semithin sections revealed glutamate decarboxylase immunopositive dots surrounding somatostatin-containing cell sections in the rat periventricular hypothalamic area. Up to 12 appositions were observed per cell section with an average number of 2-3 and a unimodal distribution. At the electron microscopical level pre-embedding staining of glutamate decarboxylase showed that most immunoreactive elements consisted of immunolabelled axonal endings. Most of these glutamate decarboxylase immunopositive boutons were found within the neuropil where they frequently made synapses on unidentified dendrites. Some of them were apposed to somatostatin-containing cell bodies that were identified according to the presence of immunolabelled granules using combined immunogold post-embedding staining. In many instances glutamate decarboxylase immunoreactive endings were also found to be involved in synaptic contact with somatostatin-labelled perikarya, or neuronal processes. These contacts provide the morphological basis for a direct GABAergic control of the somatostatin-containing cells regulating the secretion of growth hormone.

Taurine biosynthesis in rat brain: a new specific and sensitive microassay of cysteine sulfinate decarboxylase (CSDI) activity through selective immunotrapping and its use for distribution studies.

Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, has been shown to exist in two forms in rat brain, respectively CSDI and CSDII, one of which (CSDII) is considered to be in fact glutamate decarboxylase (GAD). CSDI assay after immunotrapping was made possible by using an anti-CSD antiserum raised in sheep immunized with a partially purified CSD fraction from liver. This antiserum immunoprecipitated both liver CSD and brain CSDI activities with the same affinity but did not inhibit their enzymatic activities. The immunotrapping of CSDI was selective without any contamination by GAD/CSDII activity. The immunotrapped CSD activity, which corresponded exactly to the amount of CSD not precipitated by a GAD/CSDII antiserum, was not inhibited by a specific irreversible GAD inhibitor. A quantitative, selective and sensitive assay was thus developed by measuring CSD activity on the solid phase after immunotrapping. Kinetic parameters of the immunotrapped enzyme remained unchanged. CSDI activity represented only a fraction, around 20% with saturating concentration of substrate, of the total CSD activity in rat brain homogenate. This indicates that most studies on total CSD activity dealt essentially with CSDII activity that is indeed GAD. Regional and subcellular distributions of CSDI have been determined. CSDI activity was about threefold higher in the richest (cerebellum) compared to the poorest (striatum) region without any correlation with GAD/CSDII distribution. Subcellular distribution showed a fourfold enrichment of CSDI activity in the synaptosomal fraction. The precise role of CSDI and CSDII in the biosynthesis of taurine in vivo remains to be elucidated.

Monoclonal antibodies against rat brain glutamic acid decarboxylase (GAD).

Monoclonal antibodies against rat brain GAD have been produced and immunochemically characterized in comparison with a traditional anti-GAD antiserum (Oertel et al., Neuroscience6, 2689-2700, 1981). An immunopurified fraction in which GAD represented an estimated 5% of the total protein was used as immunogen. Out of 10 mice injected with this fraction, 6 appeared to be immunized: their sera immunoprecipitated quantitatively GAD activity. Three cell fusions were performed between spleen cells of the best immunized mice and SP2/OAg14 myeloma cells. Around 500 hybridoma were generated in each hybridization experiment. The culture medium of 13 hybridoma significantly trapped GAD activity. All immunoprecipitation curves established with the ascitic fluid obtained from the positive hybridoma, showed a lower titer, at least 50-fold, than the titer of the conventional antiserum. None of these ascitic fluids was able to stain directly any protein from a rat high speed supernatant after western blotting. However, the electrophoretical analysis of the proteins immunotrapped by any of the monoclonal antibodies, followed by western blotting and immunolabelling with the anti-GAD antiserum ("cross-immunoblotting") showed the same two stained monomers. They have the same molecular weight (respectively 59 and 62 kDa +/- 2 kDa) as those stained directly by the anti-GAD antiserum from a rat brain supernatant. Although all monoclonal antibodies showed a lower affinity then the conventional antiserum, which prevents them from being used directly in immunoblotting they permit to definitively establish that the two monomers immunolabelled by the conventional antiserum are constitutive subunits of the rat brain GAD.

Non-Purkinje cell GABAergic innervation of the deep cerebellar nuclei: a quantitative immunocytochemical study in C57BL and in Purkinje cell degeneration mutant mice.

Purkinje cell degeneration (pcd) mutant mice, 3-4 months old, were used to identify and quantify the non-Purkinje cell GABAergic innervation of deep cerebellar nuclei. Glutamic acid decarboxylase (GAD) immunoreactive structures appeared as dark dots throughout the 4 nuclei. Ultrastructural examination confirmed that each dot corresponded to an axon terminal. GAD-labeled boutons were large, contained tightly packed flattened vesicles and established Gray type II synapses with all nuclear neuronal populations. Thus, cytological criteria did not distinguish between Purkinje cell and non-Purkinje cell GAD-positive nerve terminals, since they shared many common features. The number of GAD-immunoreactive axon terminals in the deep nuclei of pcd cerebella was compared to that of normal C57BL mice. Despite an almost complete disappearance of Purkinje cells in the pcd mouse (less than 0.05% of these neurons remained in the mutants), the surface density of GAD-positive nerve terminals in the deep nuclear region was 37% of control value. Taking into account a volumetric decrease of 58% for the deep nuclei of the mutant cerebellum, we estimated the percentage of GAD-positive boutons innervating these nuclei to be 15% of normal values. This important residual innervation of the deep nuclei might arise from local GABAergic neurons, which were identified in the normal and mutant cerebella by immunostaining with an anti-GABA antibody.

Decreased GABAergic innervation of the pituitary intermediate lobe after rostral hypothalamic cuts.

The effects of hypothalamic cuts at various rostro-caudal levels on the GABAergic innervation of the neurointermediate lobe of the pituitary gland have been studied. The GABAergic innervation was visualized through glutamate-decarboxylase (GAD) immunocytochemistry. Caudal hypothalamic cuts which transected the pituitary stalk completely abolished the GAD immunoreactive plexus. Rostral cuts which separated about one-third of the median eminence and arcuate nucleus from the pituitary gland decreased the GAD-immunoreactive network in the intermediate lobe but did not affect the neural lobe significantly. Although the precise location of the cell bodies giving rise to the GABAergic innervation of the neurointermediate lobe remains unknown, our findings indicate that their projections are descending ones. They are severed by rostral hypothalamic cuts and show a rostrocaudal arrangement. It is likely that the GABAergic endings of the intermediate lobe originate in the rostral hypothalamus, probably in the rostral part of the arcuate nucleus and/or in the anterior periventricular area. The GABAergic fibers in the neural lobe have a more caudal origin than those innervating the intermediate lobe.

GABA-ergic control of alpha-melanocyte-stimulating hormone (alpha-MSH) release by frog neurointermediate lobe in vitro.

Measurement of glutamate decarboxylase (GAD) activity in the intermediate lobe of the frog pituitary and brain showed that neurointermediate lobe extracts represented 12% of the GAD activity detected in the whole brain. No significant activity was measured in distal lobe extracts. Immunocytochemical studies revealed GAD-containing fibers among the parenchymal cells of the pars intermedia. The localization of GAD-like material in the intermediate lobe of the frog pituitary suggested a possible role of gamma-aminobutyric acid (GABA) in the regulation of melanotropic cell secretion. Administration of GABA (10(-6) to 10(-4) M), to perifused neurointermediate lobes caused a brief stimulation of alpha-melanocyte stimulating hormone (alpha-MSH) release followed by an inhibition. Picrotoxin (10(-4) M), a Cl- channel blocker, abolished only the stimulatory effect of GABA (10(-4) M), whereas bicuculline (10(-4) M), a specific antagonist of GABAA receptors, totally inhibited the effects of GABA (both stimulatory and inhibitory phases). Bicuculline induced by itself a slight stimulation of alpha-MSH release, suggesting that GABA-ergic nerve fibers present in the intermediate lobe are functionally active in vitro. The GABAA agonist muscimol (10(-7) to 10(-4) M) mimicked the biphasic effect of GABA on alpha-MSH release. Administration of baclofen, a specific GABAB agonist (10(-7) to 10(-4) M) induced a dose-dependent inhibition of alpha-MSH secretion. In contrast to GABA or muscimol, baclofen did not cause any stimulatory effect whatever the dose. Taken together these result suggested that GABAA and GABAB receptors were present on frog melanotrophs.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical analysis of the GABAergic innervation of oxytocin- and vasopressin-secreting neurons in the rat supraoptic nucleus.

Antisera specific for gamma-aminobutyric acid (GABA) or its biosynthetic enzyme, glutamate decarboxylase, were used in pre- and postembedding immunocytochemical techniques at the light and electron microscopic levels, to visualize the GABAergic innervation of the hypothalamic supraoptic nucleus. Immunostaining for glutamate decarboxylase or gamma-aminobutyric acid were also combined with oxytocin and vasopressin immunolocalization, thereby permitting evaluation of the contribution of the innervation onto each type of neuron in this nucleus. Light microscopy of semithin plastic sections or vibratome slices stained for glutamate decarboxylase or gamma-aminobutyric acid, with peroxidase-antiperoxidase as immunolabel, revealed an extensive punctate labeling in the supraoptic nucleus and its immediate surroundings. Quantitative analysis of glutamate decarboxylase immunostaining in semithin sections indicated a comparable density of immunopositive punctae at the anterior and posterior levels of the nucleus (14-27 X 10(6) per mm3 tissue). Glutamate decarboxylase- or gamma-aminobutyric acid-immunoreactive cell bodies were never observed within the nucleus although they were detected in the hypothalamus immediately dorsolateral to the nucleus. Electron microscopy of vibratome slices treated with antiglutamate decarboxylase or antigamma-aminobutyric acid and peroxidase-antiperoxidase, or of ultrathin sections stained directly with antigamma-aminobutyric acid and immunoglobulin-coupled colloidal gold, showed that the immuno-reactive punctae represented, in the main, axonal terminals. They invariably contained small, rounded clear vesicles and, at times, one or two larger, dense cored vesicles; they all formed symmetrical synapses onto magnocellular cell bodies and dendrites. Oxytocin and vasopressin neurons were contacted in a similar fashion by glutamate decarboxylase- or gamma-aminobutyric acid-positive boutons in semithin sections of the nucleus stained simultaneously for glutamate decarboxylase and oxytocin and in ultrathin sections stained for glutamate decarboxylase or gamma-aminobutyric acid and oxytocin or vasopressin. Glutamate decarboxylase- or gamma-aminobutyric acid-positive terminals often formed synapses onto two postsynaptic elements in the same plane of section ("double" synapses), a synaptic configuration usually encountered in supraoptic nuclei of lactating animals. In such cases, the postsynaptic somata were oxytocinergic.(ABSTRACT TRUNCATED AT 400 WORDS)

Phylogenesis of brain glutamic acid decarboxylase from vertebrates: immunochemical studies.

Brain high-speed supernatants from various lower and higher vertebrates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblot on nitrocellulose membranes, and immunolabelling using an anti-glutamic acid decarboxylase (anti-GAD) antiserum prepared from rat antigen. Rat brain extracts showed two distinct immunolabelled bands (MW 59,000 and 62,000 daltons). The molecular weight of the native enzyme was 120,000 daltons. The immunoblot pattern was not affected by a 3-h incubation of the homogenate. In the substantia nigra, the decrease in the immunolabelling of both bands corresponded very closely to the decrease of GAD activity following lesioning of the striato-nigral pathway. Moreover, experiments with preadsorbed antiserum showed that both subunits have common antigenic determinants. The immunolabelling was consistently more intense over the lightest band. The autoradiography of immunoprecipitated rat brain GAD, iodinated prior to electrophoresis, revealed two radiolabelled bands corresponding to the two immunolabelled ones. Their radioactivity was found in a one-to-five ratio which closely paralleled their respective immunolabelling intensity. Thus, the two subunits recognized by the antiserum are not present in stoichiometric proportions in the rat brain high-speed supernatant. These findings suggest the existence of two homodimeric GAD with common antigenic determinants which are present in different amounts. Immunoprecipitation curves of brain GAD from rat, mouse, rabbit, monkey, human, quail, frog, and trout were similar, with a less than 10-fold maximum shift in affinity for GAD. GAD immunoblots from the various higher vertebrates showed a pattern similar to that obtained in rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate decarboxylase-immunoreactive boutons in synaptic contacts with hypothalamic dopaminergic cells: a light and electron microscopy study combining immunocytochemistry and radioautography.

Double post-embedding immunolabeling of both tyrosine hydroxylase and glutamate decarboxylase on 1-micron semi-thin sections allowed the visualization of numerous endings that use gamma-aminobutyrate as a transmitter apposed to dopaminergic cell bodies in the periventricular-arcuate hypothalamic complex. Up to fifteen glutamate decarboxylase-positive contacts per tyrosine hydroxylase-positive cell profile could be observed. In some favourable planes of section glutamate decarboxylase-positive endings were also seen in close apposition to proximal dopaminergic dendrites. About 250 tyrosine hydroxylase-positive cell profiles, whose diameter approached the maximum diameter of the dopaminergic cells, were surveyed. An average of 7.4 glutamate decarboxylase-positive contacts were counted on these profiles. From these figures it was estimated that a dopaminergic cell body was contacted on average by 75-175 terminals that use gamma-aminobutyrate as a transmitter. At the electron-microscopic level, the nature of these contacts was investigated by a method combining radioautographic detection of cell bodies having taken up tritiated dopamine and pre-embedding immunostaining of glutamate decarboxylase containing endings. Glutamate decarboxylase-positive axon terminals were seen apposed to somatic and dendritic elements. On some favorable planes of section, they were found to be engaged in morphologically defined synaptic complexes of the symmetrical or asymmetrical type. A number of the postsynaptic perikarya were labelled by tritiated dopamine and, in agreement with the light microscopic observations, they were frequently seen in contact with more than one immunopositive ending. The present findings provide a morphological substratum for a direct gamma-aminobutyrate control of the tuberoinfundibular dopaminergic neurons. Such a control could account more particularly for the central, stimulatory effects of gamma-aminobutyrate on prolactin secretion.

Rate of tryptophan hydroxylation in vivo in brain nuclei of genetically hypertensive rats of the Lyon strain.

The rate of tryptophan hydroxylation in vivo is unaltered in brain areas of 5, 9 and 21 week-old Lyon genetically Hypertensive (LH) rats as compared to both Lyon Normotensive (LN) and Low Blood Pressure (LL) rats, except for a decrease in the C1 area of the medulla oblongata in 9 week-old animals.

Biochemical and immunochemical studies on the GABAergic system in the rat fallopian tube and ovary.

gamma-Aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) activities were measured in the ovary and the Fallopian tube of rats and compared with brain values. GABA levels in the Fallopian tube were about twice as high as in the brain, while in the ovary they represented only about 5% of the amino acid content of the CNS. In vitro decarboxylation of glutamate, measured via CO2 formation, occurred both in the Fallopian tube and in the ovary. These two organs contained, respectively, 10% and 1% of brain GAD activity. However, the actual formation of GABA from glutamate in a high-speed supernatant was detectable only in the Fallopian tube, where it represented about 5% of brain GAD activity. In contrast with the enzyme present in ovary, liver, anterior pituitary, and kidney, that in the Fallopian tube was quantitatively precipitated by a specific antiserum directed against rat neuronal GAD. Moreover, subcutaneous transplantation resulted in a quantitative decrease of both GABA levels and GAD activity in the Fallopian tube while no change occurred in the ovary, and vagus nerve section induced a 50% decrease of GAD activity in the Fallopian tube, although GABA levels were not significantly altered. The findings suggest an extrinsic GABAergic innervation in the rat Fallopian tube but not in the ovary.