A triple ultrastructural immunogold staining method. Application to the simultaneous demonstration of three hypophyseal hormones.

The triple ultrastructural immunocytochemical labeling technique described here is based on the use of three different antisera raised in two different animal species: rabbit anti-corticotropin (R-ACTH), guinea pig anti-prolactin (GP-LTH) and rabbit anti-gonadotropin (R-LH beta). Staining is carried out on both sides (A and B) of the same tissue section. First, side A is incubated with a mixture of R-ACTH and GP-LTH and then with a mixture of the two corresponding species-specific immunoglobulins (IgG) adsorbed respectively to 5 and 20 nm gold particles: goat (G) anti-R IgG 5 + G anti-GP IgG 20; second, side B is incubated with R-LH beta, followed by species-specific secondary antibodies adsorbed in 10 nm gold particles; G anti-R IgG 10. With this technique, we demonstrated, on the same thin section, ACTH, LTH, and LH cells. The immunocytochemical procedure used has proved useful for simultaneous ultrastructural localization, on the same thin section, of three different antigenic sites. This technique, applied to other materials, could provide interesting information in several biological fields.

A double-immunostaining procedure using colloidal gold, ferritin, and peroxidase as markers for simultaneous detection of two hypophysial hormones with the electron microscope.

For simultaneous ultrastructural localization of intracellular peptides, protein A-gold techniques or immuno-gold techniques have generally been applied. The present study reports a double immunostaining procedure for simultaneous visualization of two hypophysial hormones (prolactin and corticotropin) on a single ultrathin section of the pars distalis of an amphibian. Prolactin and corticotropin antisera were respectively raised in guinea pigs and rabbits and were applied simultaneously to ultrathin sections. Antigenic binding sites were detected under the electron microscope using differently labeled species-specific secondary antisera raised in goats or sheep. Three labels (gold particles, ferritin, peroxidase) were checked for double labeling. The combinations investigated were: 1) two gold preparations or IgG-gold labeled with different-sized gold particles; 2)IgG-gold and IgG-ferritin; 3) IgG-gold and IgG-PAP (peroxidase-antiperoxidase). The double-immunostaining procedures described here have proved useful in the simultaneous ultrastructural localizations of two intracellular antigens on a single tissue section. These procedures constitute a basis for the development of triple immunostaining methods.

Gastrin-like peptides in the amphibian brain: an immunohistochemical study.

Peptides homologous to the gastro-intestinal hormones, gastrin and cholecystokinin (CCK), are present in the brain. Using immunohistochemistry we localized a neurosecretory system, reacting with antigastrin sera, in the brain of amphibians by light and electron microscopy. It has a dual location: (1) in the nucleus infundibularis ventralis (NIV) two groups of perikarya give rise to fibre tracts which join up in the infundibular floor and end in the external zone of the median eminence; (2) in the preoptic area nerve fibres and endings are found near the preoptic recess. At the electron microscope level, the immuno-colloidal-gold method revealed cells characterized by small immunoreactive dense granules, localized in the perikarya, nerve fibres and endings. Specificity controls showed that the immunoreacting substance(s) contain(s) all or part of the sequence (Trp-Met-Asp-Phe) common to gastrin, CCK and pentagastrin. The exact nature(s) and physiological function(s) of these substance(s) has (have) still to be defined.

A morphological and biochemical study of the myelin-like membrane structures formed in cultures of pure oligodendrocytes.

This study reports the production of myelin-like membranes in oligodendrocyte subcultures derived from 20-day-old primary glial cell cultures of newborn rat brain. These multi-layered structures show a variable number of membrane turns; up to 10 concentric lamellae are found in 3- to 4-week-old subcultures. When they are compacted, alternate dense and intraperiodic lines with a periodicity of 11.2 nm are noticeable. The most typical myelin proteins were detected straight on the multi-lammellar structures by a gold immunocytochemical method. Subcellular fractions containing these myelin-like structures were isolated by ultracentrifugation on a discontinuous sucrose gradient. They were analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting; UDP-galactose: ceramide galactosyltransferase and 2',3'-cyclic nucleotide 3'-phosphohydrolase activities were also measured. The results indicate that the multi-layered membrane profiles have many characteristics of the myelin found in vivo; nevertheless some differences were still apparent. Our data support the concept of the cultured oligodendrocytes expressing the intrinsic myelinogenic properties and possessing a basic developmental program of myelination, apparently in the absence of stimuli coming from other brain cells.

Immunohistochemical study on the development of the adenohypophysial cells in the lizard Gallotia galloti.

Immunohistochemical methods have been used to study the embryonic and postnatal development of the hormone-producing cells in the adenohypophysis of the lizard Gallotia galloti. In this species, Rathke's pouch is formed between stages 30 to 32 of the embryonic development, although the first sign of immunoreactivity to antisera against adenohypophysial hormones occurs in stage 33 in the pars distalis anlage. These cells derive from the dorsal face of Rathke's pouch and are immunoreactive to anti-ACTH serum. The cytodifferentiation of ACTH and MSH cells occurs in the pars intermedia in stage 34. The TSH cells appear at stage 35 and the gonadotrope cells at stage 37. These cells derive from both the dorsal and ventral face of the Rathke's pouch. The LTH cells are revealed at stage 39 and are only originated from the dorsal face. The STH cells, which come from the dorsal as well as ventral face, are the last secretory cells differentiated just before hatching. During postnatal development an increase and also a redistribution of the immunoreactive cells occur until acquiring the adult distribution.

Gerstmann-Sträussler-Scheinker disease with A117V mutation in a second French-Alsatian family.

We report a kindred of French/Alsatian origin with symptoms of Gerstmann-Sträussler-Scheinker disease over 3 generations. In the propositus, cerebellar signs and memory disturbance were the presenting features, followed by other neurological manifestations. Biopsy of the cerebral cortex showed numerous multicentric and "kuru"-type amyloid plaques that on immuno-light and electron microscopy stained with antibody to prion protein. Molecular genetic analysis revealed an A117V mutation in the open reading frame of the prion protein gene. Questions as to pathology and spread of this mutation are discussed.

[Creutzfeldt-Jakob disease after treatment with human extracted growth hormone. A clinicopathological study]. / Maladie de Creutzfeldt-Jakob après traitement par l'hormone de croissance extractive humaine: étude clinico-pathologique.

This report describes the pathological changes observed in the brain of a 18-year-old patient who died with Creutzfeldt-Jakob disease (CJD) of the ataxic and panencephalopathic type. Clinically, the disease began at age 17 with a rapidly progressive cerebellar syndrome, associated with myoclonus and mental deterioration. Cranial CT scan and MRI showed minor abnormalities. EEG demonstrated non specific changes. The patient became progressively demented, bedridden and died 10 months after the onset. Nine years earlier, he had been operated upon for craniopharyngioma and subsequently treated with cadaver-derived human growth hormone. Post-mortem examination of the brain revealed mild diffuse atrophy. Histology showed congophilic amyloid plaques found in both gray and white matters of the cerebrum and cerebellum, associated with spongiosis, mainly in the basal ganglia and cerebellum, and gliosis. They were immunostained with antiprion protein antibody using the immunoperoxidase method on paraffin embedded sections. The clinical findings in this case were similar to those of other cases of human growth hormone-associated CJD. However, this case is unusual because of the great number of amyloid plaques, which have been only rarely found in previous reports.

Immunohistochemical detection of adenohypophyseal cells containing hormones in Rana ridibunda.

The pars distalis of the pituitary of Rana ridibunda captured throughout the spring and summer was examined with immunofluorescence techniques using antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, four different cell types of recognized: 1) cells immunoreactive to anti-bovine LTH, 2) cells immunoreactive to anti-bovine STH, 3) cells immunoreactive to anti-ovine LH, and 4) cells immunoreactive to anti-synthetic ACTH beta (1-24). Their distribution and morphology as well as their staining characteristics (classical histological techniques) are reported in this study.

The monoaminergic system in the diencephalon of the newt tadpole, Triturus alpestris (Mert). A histofluorescence study.

The distribution of monoamines in the brain of Triturus alpestris tadpoles was investigated with the Falck-Hillarp histofluorescence method. This study concerns especially the diencephalon where is observed the major part of the monoaminergic (MA) neurons. MA fiber tracts are found in the striatum, the septal nucleus and the medial and lateral forebrain bundles of the telencephalon. In the anterior diencephalon cerebrospinal fluid (CSF)--contacting catecholaminergic cells are localized within the preoptic recess organ (PRO) and fibers in the preoptic nucleus. More caudally both CSF-contacting catecholaminergic and serotonergic cells can be demonstrated in the paraventricular organ (PVO) and in the nucleus infundibularis dorsalis (NID). In the hypothalamo-hypophysial tract, numerous catecholaminergic (CA) fibers occur in the median eminence and in the pars intermedia. The origin of these fibers is not elucidated but a participation of the PRO, PVO and NID seems possible. CA cells without contact with the CSF are localized laterally to the PVO, PVO-accompanying cells, and in the nucleus reticularis mesencephali (NRM). The distribution of MA neurons in the brain of Triturus alpestris appears more similar to that of anurans than that of the primitive urodeles.

LHRH-like system in the brain of Xenopus laevis Daud: immunohistochemical idenfication.

Rabbit antiserum to synthetic LHRH was used with the immunofluorescence technique to identify the LHRH-secreting neurons and their axonal pathways in the brain of Xenopus laevis. Three groups of immunoreactive neurons were identified: the first, in the telencephalon, is a paired group of cells scattered near the two telencephalic ventricles; the second group lies near the preoptic recess; the third group occurs in the ventral wall of the infundibulum. Two principal neuronal pathways were observed: Fibres originating from the dorsally located telencephalic neurons converge on the cephalic median plane where they form a single bundle behind the telencephalic furrow. This bundle descends towards the anterior border of the preoptic recess where it divides into two nerve bundles which pass on either side of the preoptic recess, run above the optic chiasma then cross the infundibular floor and finally terminate in the median eminence. The second pathway is more direct. The more ventrally located telencephalic LHRH cells give rise to this second pathway. Their axons converge with the other LHRH fibres near the lateral border of the preoptic recess. Most of the LHRH nerve fibres terminate in the median eminence although some terminate near the paired pars tuberalis. No reaction was observed after the use of antiserum absorbed with synthetic antigen.

Immunohistochemical study of the adenohypophysis of Typhlonectes compressicaudus (Amphibia, Gymnophiona).

The indirect immunofluorescence method was used to identify and locate LTH-, STH-, LH-, TSH-, ACTH- and MSH-immunoreactive cells in the pituitary of Typhlonectes compressicaudus (Gymnophiona). The present study defines the histological and histochemical staining properties of each cell type identified.

[Immunofluorescent localization of pituitary hormones during embryonic development of Nectophrynoides occidentalis Angel]. / Localisation par immunofluorescence de différents principes hormonaux de l'hypophyse de Nectophrynoïdes occidentalis angel, au cours du développement embryonnaire.

From premetamorphosis (end of stage Ib) of Nectophrynoïdes occidentalis (viviparous anuran amphibian) MSH cells are revealed in the pars intermedia of pituitary using cytoimmunofluorescence technique. At the same stage, ACTH-STH-PRL and LH cells are revealed in the pars distalis. The comparison of the order of appearance of different cell categories in the adenohypophysis (considered in the functional point of view) among the embryos of Nectophrynoïdes and the tadpoles of different amphibians allows the emphasis of some analogies and divergences.

Miniplaques and shapeless cerebral amyloid deposits in a case of Gerstmann-Sträussler-Scheinker's syndrome.

Multicentric plaques of typical pathomorphology have been described in two members of the well-documented German family "Sch" with Gerstmann-Sträussler-Scheinker's syndrome (GSS). The case of a third affected member with a clinical course of 10 years is now reported. In addition to multicentric plaques, small granules of prion protein (PrP) with stellate ultrastructure (miniplaques) were found in the tractus perforans and alveus of the hippocampal area. At the ultrastructural level, shapeless amyloid deposits of irregularly arranged bundles of amyloid fibrils were also observed in the same region. All amyloid precipitates apparent at the light microscopic level immunostained for PrP. These observations widen the known spectrum of amyloid plaque morphology in GSS. The multicentric plaque should still, however, be considered the diagnostic hallmark of this disease.

[Localization and identification of the LH-RF center in the brain of the toad, Bufo vulgaris Laur]. / Localisation et identification d'un centre LH-RF dans l'encéphale du Crapaud, Fu vulgaris Laur

Using indirect immunofluorescence procedure with an anti-synthetic LHRF serum, pericarya of neurons producing LHRF or an LHRF-like substance were revealed in hypophysectomized Bufo vulgaris. They are localized principally in the forebrain near the posterior end of the first and second ventricles. The fluorescent axons coming from these pericarya are visible on their pathway to a region near tha pars tuberalis and to the median eminence.

Immunohistochemical localisation of substances reactive to antisera against alpha- and beta-endorphin and met-enkephalin in the brain of Rana temporaria L.

In the brain of Rana temporaria, two distinct systems reactive with alpha- and beta-endorphin antisera, respectively, and with a met-enkephalin antiserum, have been detected immunohistochemically. Neurons reacting with alpha- and beta- endorphin antisera are located (1) in the preoptic nucleus, and (2) in the pars ventralis of the tuber cinereum. Immunoreactive nerve fibres of both groups of perikarya end in the infundibular floor near the capillaries and the preoptico-hypophysial tract. Control reactions have shown that the immunoreactivity is suppressed by the corresponding antigens, but also by beta-LPH. In view of these results the immunoreactive systems examined correspond to an alpha/beta-endorphin system or a lipotropinergic system. neurons reacting with the met-enkephalin antiserum are located in the paraventricular organ. Intense immunofluorescence was observed in the infundibular floor. Controls show that the labelling by met-enkephalin antiserum is exclusively suppressed by met-enkephalin. In the pituitary gland, on the other hand, alpha- and beta-endorphin antisera reveal: 1) the MSH/ACTH-like cells of the pars intermedia and 2) the ACTH-like cells of the pars distalis.

Immunohistochemical demonstration of TSH-, LH- and ACTH-cells in the hypophysis of tadpoles of Xenopus laevis D.

By use of the immunofluorescence technique TSH-, LH- and ACTH-cells were localized in the hypophysis of tadpoles of Xenopus laevis. The first signs of the activity of these cells were observed in early stages of the development, i.e., stage 39 for ACTH, and stage 42 for TSH and LH.

[Immunofluorencence localization of secretions related to gonadotropic, corticotropic, melanotropic and somatotropic hormones in hypophysis of Nectophrynoides occidentalis]. / Localisation par immunofluorescence des sécrétions apparentées aux hormones gonadotrope, corticotrope, mélanotrope et somatotrope dans l'hypophyse de Nectrophrynoïdes occidentalis

In Nectophrynoïdes occidentalis, ovine anti-LH anti beta (1-24) corticotropin, both anti alpha and anti beta MSH and bovine anti-STH antisera allowed us to identify by immunofluorescence gonadotropic, corticotropic, melanotropic and somatotropic hypophysial cells of a viviparous toad. The identification of these different cell types was confirmed by applying a classical cytologic technique to the same sections.

[Immunohistochemical identification, in an anuran amphibian (Xenopus laevis Daud.) of infundibular neurons reacting with antigastrin serum]. / Identification immunohistochimique, chez un Amphibien anoure (Xenopus laevis Daud.) de neurones infundibulaires réagissant avec un sérum anti-gastrine.

Neurons containing peptides immunocytologically related to gastrin have been revealed by immuno-cytology in the post-chiasmatic hypothalamus of Xenopus laevis. Their pericarya are localized in the postero-dorsal zone of the pars ventralis of the tuber cinereum; their axons end on the capillaries of the external zone of the median eminence.