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.

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.

Anatomical relationships of dopaminergic and GABAergic systems with the GnRH-systems in the septo-hypothalamic area. Immunohistochemical studies.

Immunohistochemical double staining for gonadotropin releasing hormone (GnRH) and tyrosine hydroxylase (TH) or glutamic acid decarboxylase (GAD) reveals in the septo-preoptic-diagonal band complex of the rat brain close spatial associations between GnRH-immunoreactive perikarya and TH and GAD immunoreactive fibers. In the organum vasculosum laminae terminalis, no close spatial relationships could be observed between TH- or GAD-positive fibers and the GnRH-containing system. In contrast, in the median eminence substantial overlap exists in the distribution of GnRH with TH and GAD containing nerve fibers. This overlap is most intense for TH throughout the lateral palisade zone, while for GAD it is more restricted to the outermost portion of the external palisade zone. The results suggest that dopamine and GABA influence GnRH secretion via axosomatic contacts in the septo-preoptic-diagonal band complex, as well as via axo-axonic interactions in the median eminence, while no such interactions seem to exist in the organum vasculosum laminae terminalis. Since dopaminergic cell bodies in the ventral hypothalamus are closely apposed by GnRH and GAD containing fibers, the existence of feedback circuits among GnRH, dopamine and GABA systems is proposed.

GABAergic neurons in dissociated cultures of rat hypothalamus, septum, and midbrain.

Cultures of dissociated tissue blocks from 18 day-old rat embryos containing either septum plus anterior hypothalamus, retrochiasmatic hypothalamus, or midbrain, were maintained for 8-29 days in vitro. GABAergic neurons were visualized by incubation with (3H) gamma-aminobutyric acid followed by autoradiography and by immunocytochemistry using an antiserum against glutamic acid decarboxylase. Positive neurons were of variable shapes and sizes. Labeled neurons in the three cultured regions represented 3.4, 1.1 and 1.7%, respectively, of all neurons. Apart from a few exceptions, autoradiographic labeling and immunocytochemical staining coincided. Glial cells did not stain and showed very low grain densities. Radioactivity could be released from preloaded cultures upon depolarization by 50 mM potassium and 100 microM Veratridine. The demonstration of GABAergic neurons in cultures that are known to contain a variety of peptidergic neurons opens up the possibility of studying the interaction between the two classes of neurons in vitro.

Origin of glutamate-decarboxylase (GAD)-containing cells in discrete hypothalamic nuclei.

Glutamate decarboxylase activity (GAD) was assayed in discrete hypothalamic nuclei in the rat following lesions of the major afferent pathways from hippocampus, amygdala, midbrain, septum, thalamus and globus pallidus. None of these lesions led to a marked decrease in GAD activity in selected nuclei. After total deafferentation of the medial-basal hypothalamus GAD remained unchanged in the median eminence but fell markedly in the ventromedial and arcuate nuclei. In these two nuclei a decrease of GAD still occurred following partial deafferentation from lateral and posterior hypothalamus, but not from anterior and preoptic areas. These results indicated that most GAD-containing cells have their origin inside of the hypothalamus. In this region GABAergic neurons are likely to be short interneurons providing intrahypothalamic connections. Such connections are suggested from lateral and posterior hypothalamus onto the medial basal nuclei.

Glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in discrete nuclei of hypothalamus and substantia nigra.

The distribution of L-glutamate decarboxylase activity (GAD) and gamma-aminobutyric acid (GABA) was investigated in the hypothalamic nuclei and in parts of the substantia nigra in the rat. GAD varied markedly among these areas. The reticular part of the nigra showed the highest activity two-fold higher than any other nucleus. Among the hypothalamic nuclei, a 5-fold difference was found between the poorest and richest nuclei. High GAD was measured in the preoptic, anterior and dorsomedial nuclei. Low activity was found in arcuate and supraoptic nuclei. The lowest GAD activity was measured in the median eminence with only half the activity of the whole brain homogenate. This suggests that GABAergic neurones might not be involved in neuroendocrine regulation at the median eminence level. GABA was determined using the sensitive cycling microassay. The rats were killed by microwaves, procedure which was found to inactivate enzymatic processes within two sec without affecting the level or the distribution of GABA. Postmortem increments during the first 3 min following decapitation ranged up to 5 times the endogenous levels, and were proportional to the GAD activity of the corresponding nucleus. This confirms that GAD is the limiting factor in GABA synthesis and suggests that GABA turnover might be rapid. Endogenous GABA showed a uniform distribution within hypothalamic nuclei and nigra. No relationship appeared between endogenous GABA levels and GAD activities in the various nuclei. These results suggest that while GABA synthesis is likely to occur in non-evenly distributed nerve cells, most GABA may be stored in surrounding cells, presumably glia cells.

Effect of surgical isolation of the hypothalamus on its neurotransmitter content.

The concentrations of norepinephrine, dopamine-beta-hydroxylase, dopamine, tyrosine hydroxylase, phenylethanolamine-N-methyltransferase, serotonin, tryptophan hydroxylase, histamine, glutamic acid decarboxylase, and choline acetyltransferase were determined in selected hypothalamic nuclei and in the median eminence after deafferentation of the medial basal hypothalamus. Norepinephrine and dopamine-beta-hydroxylase fell markedly while dopamine and tyrosine hydroxylase did not. Serotonin also decreased in all regions studied; histamine decreased in none. Choline acetyltransferase, phenylethanolamine-N-methyltransferase, and glutamic acid decarboxylase declined in some areas, but not in others.