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.

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)

GABAergic regulation of melanocyte-stimulating hormone secretion from the pars intermedia of Xenopus laevis: immunocytochemical and physiological evidence.

alpha-MSH secretion from the amphibian pars intermedia is under inhibitory hypothalamic control, and the catecholamine dopamine is thought to be the physiological MSH release-inhibiting factor. In the present study we evaluated the possible role of the neurotransmitter gamma-aminobutyric acid (GABA) in the regulation of the pars intermedia of Xenopus laevis. Immunocytochemical staining with antibodies to glutamic acid decarboxylase showed the presence of a rich GABAergic network in the intermediate lobe of the pituitary gland. Administration of GABA to superfused neurointermediate lobes caused a rapid and dose-dependent inhibition of basal release of MSH and immunoreactive endorphin. Pulse-chase experiments revealed that GABA gave a coordinate inhibition of the release of all peptides derived from proopiomelanocortin. In vivo administration of GABA resulted in almost complete pigment aggregation in dermal melanophores of both adults and larvae. Altogether, our results indicate that GABA is a physiologically important factor for regulation of the pars intermedia in Xenopus laevis.

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.

Catecholaminergic and GABAergic anatomical relationship in the rat substantia nigra, locus coeruleus, and hypothalamic median eminence: immunocytochemical visualization of biosynthetic enzymes on serial semithin plastic-embedded sections.

The visualization of protein antigens has been performed on semithin sections embedded in Araldite. After partial removal of the resin and a light proteolytic treatment of the tissue we were able to localize several biosynthetic enzymes: tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), and glutamic acid decarboxylase (GAD), which are, respectively, markers of catecholaminergic, adrenergic, gamma-aminobutyric acid (GABA)ergic systems. This technique afforded a high resolution of light microscopy details and immunostaining of TH, GAD, and PNMT on serial sections enabled us to compare with great precision GABAergic and adrenergic innervations in the rat locus coeruleus. In addition, it allows us to study the possible relationship between these terminals and the noradrenergic neurons. We also compared the general pattern of distribution of TH- and GAD-positive endings in the hypothalamic median eminence. The preliminary results obtained with this technique revealed some interesting facts previously unseen when preparations with lower histological resolution were used.

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.

Absence of light-dark entrainment on the sleep-waking cycle in mice with intact visual perception.

The influence of the light-dark schedule (12 h-12 h) on the sleep-waking cycle has been studied in anophthalmic mice: the 'eyeless' ZRDCT/An strain. The complete anophthalmic mice or the heterozygotous mice of the same strain with unilateral or bilateral eyeballs present a circadian organization of the sleep-waking cycle which is not dependent on the light-dark cycle. These results are different from sleep rhythms of C57Br mice recorded under the same experimental conditions. They indicate that the structures responsible for the circadian rhythmicity of sleep exist in all the 'eyeless' ZRDCT/An mice, but are not functionally linked with the visual system even in the mice with unilateral and bilateral eyeballs.

[Genetic study of paradoxical sleep in mice. Connection with coloration genes]. / Etude génétique du sommeil paradoxal chez la souris. I. Liaison possible avec les gènes de coloration.

The study of Paradoxical Sleep (PS) from 94 male mice belonging to five inbred strains: C57BR/cdOrl (BR), BALB/cOrl (C), AKROrl, A/JOrl (A/J) and C57BL/6-c2J (B6-c2J), reciprocal F1 hybrids between BR and C strains and backcrosses between F1 hybrids and BR or C was carried out. The results showed that 1) night PS duration was independent from that of day PS; 2) C type of PS seemed to be dominant over BR type; 3) C type is linked to the albino gene; and 4) the albino gene, per se, is not involved in PS regulation. From these results a two locus model was elaborated. This model explains most of the results. The nature of such a genetic support was discussed.