Identification of a novel HLA allele, HLA-B*53:39, in a Guadaloupean individual.

The novel HLA-B*53:39 allele differs from HLA-B*53:01 by a single nucleotide substitution at codon 45 (ACG>AAG).

Ependymal and choroidal cells in culture: characterization and functional differentiation.

During the past 10 years, our teams developed long-term primary cultures of ependymal cells derived from ventricular walls of telencephalon and hypothalamus or choroidal cells (modified ependymal cells) derived from plexuses dissected out of fetal or newborn mouse or rat brains. Cultures were established in serum-supplemented or chemically defined media after seeding on serum-, fibronectin-, or collagen-laminin-coated plastic dishes or semipermeable inserts. To identify and characterize cell types growing in our cultures, we used morphological features provided by phase contrast, scanning, and transmission electron microscopy. We used antibodies against intermediate filament proteins (vimentin, glial fibrillary acidic protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin, and fibronectin in single or double immunostaining, and monoclonal antibodies against epitopes of ependymal or endothelial cells, to recognize ventricular wall cell types with immunological criteria. Ciliated or nonciliated ependymal cells in telencephalic cultures, tanycytes and ciliated and nonciliated ependymal cells in hypothalamic cultures always exceeded 75% of the cultured cells under the conditions used. These cells were characterized by their cell shape and epithelial organization, by their apical differentiations observed by scanning and transmission electron microscopy, and by specific markers (e.g., glial fibrillary acidic protein, ciliary rootlet proteins, DARPP 32) detected by immunofluorescence. All these cultured ependymal cell types remarkably resembled in vivo ependymocytes in terms of molecular markers and ultrastructural features. Choroidal cells were also maintained for several weeks in culture, and abundantly expressed markers were detected in both choroidal tissue and culture (Na+-K+-dependent ATPase, DARPP 32, G proteins, ANP receptors). In this review, the culture models we developed (defined in terms of biological material, media, substrates, duration, and subculturing) are also compared with those developed by other investigators during the last 10 years. Focusing on morphological and functional approaches, we have shown that these culture models were suitable to investigate and provide new insights on (1) the gap junctional communication of ependymal, choroidal, and astroglial cells in long-term primary cultures by freeze-fracture or dye transfer of Lucifer Yellow CH after intracellular microinjection; (2) some ionic channels; (3) the hormone receptors to tri-iodothyronine or atrial natriuretic peptides; (4) the regulatory effect of tri-iodothyronine on glutamine synthetase expression; (5) the endocytosis and transcytosis of proteins; and (6) the morphogenetic effects of galactosyl-ceramide. We also discuss new insights provided by recent results reported on in vitro ependymal and choroidal expressions of neuropeptide-processing enzymes and neurosecretory proteins or choroidal expression of transferrin regulated through serotoninergic activation.

Cardiac and plasma atrial natriuretic peptide after 9-day hindlimb suspension in rats.

To determine atrial natriuretic peptide (ANP) adaptation to simulated weightlessness, immunoreactive plasma (ir-NH2- and ir-COOH-terminals) and atrial (ir-COOH-terminal) ANP levels, atrial mRNA expression, immunoreactive cardiocyte ANP levels (ir-NH2- and ir-COOH-terminals), and ultrastructural observations of granules in atrial cardiocytes were assessed in male Wistar rats after a 9-day hindlimb suspension. Plasma ir-NH2- and ir-COOH-terminal ANP concentrations decreased by 17 (P < 0.05) and 37% (P < 0.05), respectively, in suspended rats. A concomitant ir-COOH-terminal ANP content reduction was also observed in left (31%; P < 0.01) and right atria (25%; P < 0.05). Atrial ANP mRNA expression was severely depleted in the right atrium and less so in the left atrium after 9 days of hindlimb suspension. Immunocytochemistry observations demonstrated lowered NH2- and COOH-terminal ANP immunoreactivities in left and right atria from suspended rats. A reduced number of storage granules (dense granules) in both atria was also noted on ultrastructural analysis. It was concluded that ANP biosynthesis, storage, and release were decreased after a 9-day hindlimb suspension.

Gap junctional intercellular communication between cultured ependymal cells, revealed by lucifer yellow CH transfer and freeze-fracture.

In order to analyze intercellular communication between ependymal cells in mammalian brain, we have studied gap junctional communication of ependymal and glial cells in long term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus obtained in serum supplemented media with two complementary methods: 1) dye transfer of Lucifer Yellow CH after intracellular microinjection of the different cellular types, and 2) freeze-fracture of the same cultured ependymal cells. In our culture conditions, we have shown that the GJIC capacity to transfer dye was very different according to cellular types microinjected with Lucifer Yellow CH in the following respects: 1) in ependymal cells, GJIC was always important: ciliated ependymal cells, which are numerous in hypothalamic ependymal cultures (10-120 coupled cells), choroidal ependymocytes in plexus cultures (15-250 coupled cells), and non-choroidal ependymocytes in diencephalic roof cultures (10-30 coupled cells), and 2) in astroglial cells found in these primary cultures, no GJIC was observed in spite of the presence of well-differentiated gap junctions revealed by freeze-fracture replicas. All these results show a strong GJIC in ependymal cells and indicate the very good functional state of these cells in vitro.

Apical localization of the alpha subunit of GTP-binding protein Go in choroidal and ciliated ependymocytes.

The presence of GTP-binding proteins (G proteins) has been studied in murine adult choroid plexuses and cultured fetal choroidal or hypothalamic ependymal cells by ADP-ribosylation catalyzed by Bordetella pertussis toxin (PTX) and by immunodetection using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha), the major brain G protein. ADP-ribosylation with 32P-NAD and PTX of choroid plexus revealed an intense labeling at the 40 kDa level in addition to the known PTX-substrates at 41 kDa (Gi alpha) and 39 kDa (Go alpha). This 40 kDa substrate was also predominant in cultured ependymal cells. However, a positive immunoreactivity with the anti-Go alpha antibodies was detected at the level of the 39 kDa faster component, indicating the presence of Go alpha in both choroid plexuses and cultured ependymal cells. In thin frozen sections as well as in cultured cells, Go alpha was mainly immunolocalized at the apical pole of choroidal ependymocytes and in the kinocilia of ciliated ependymal cells. At the ultrastructural level, using gold immunoprobes, the immunoreactivity of a Go alpha-like protein was detected on the cytoplasmic face of the apical plasma membrane, coated pits and vesicles, and in the apical cytoplasmic matrix. In ciliated ependymal cells, the positive immunostaining displayed a dotted pattern at the surface of demembranated axonema of apical kinocilia. These findings strongly suggest that G proteins, especially Go, are involved in transducing chemical signals that modulate traffic and exchanges between cerebrospinal fluid and ependyma through the apical membrane of ependymocytes.

Topographical distribution of CRF immunoreactivity in the pigeon brain.

The distribution of corticotropin-releasing factor (CRF) immunoreactivity was demonstrated by immunocytochemistry in intact and colchicine-treated pigeons. Colchicine injections were administered at different times related to the circadian activity of the CRF-adrenocorticotropin (ACTH)-corticosterone axis. Three CRF antisera were used, two directed against synthetic rat CRF and one directed against synthetic ovine CRF. No fundamental differences appeared in the pigeon brain with respect to the specific CRF antiserum used. The most effective colchicine injection times corresponded to hypersecretion in the corticotropic axis. CRF-immunopositive neurons were scattered throughout the pigeon brain. In addition to the paraventricular hypothalamic system, which is involved in adenohypophysial ACTH regulation, several other hypothalamic and extrahypothalamic areas showed CRF neurons. The distribution suggests that CRF may also act as a modulator and a neurotransmitter. Two hypothalamic paraventricular nucleus-median eminence CRF pathways are described here. Moreover, CRF-immunopositive reactions were observed in specific areas of cerebral ventricle walls, suggesting that CRF may be released into the cerebral fluid.

[Immunofluorescence localization of corticoliberin neurons in the brain of pigeons]. / Localisation par immunofluorescence des neurones à corticolibérine dans l'encéphale de pigeon.

With immunofluorescence techniques using one anti-rat or two different anti-ovine CRF, the localization of corticotropin-releasing factor (CRF) producing neurons was characterized in frozen sections of pigeon brain. Colchicine was administered intraventricularly at various day hours. The CRF neurons were localized in the telencephalon: lobus parolfactorius, nucleus (n.) accumbens, anterior commissure; in the diencephalon: n. dorso-medialis and lateralis thalami and in different structures of the hypothalamus: n. praeopticus periventricularis and medialis, paraventricularis, supraopticus medialis, lateralis, ectomamillaris and in the stratum cellulare externum. Concerning the hypothalamic localizations, results are discussed in the light of physiological studies on corticotropic regulations in pigeons. Additional populations of CRF neurons were also located in various brainstem areas substantia grisea centralis, locus caeruleus, n. tegmenti dorsalis, sensorius principalis nervi trigemini, vestibularis latetalis, solitarius, nervi hypoglossi, in the dorsal area of the n. pontis lateralis and in the n. paramedianus paragiganto--cellularis, raphes, nervi facialis, subcaeruleus and the area ventralis. These particular localizations may lead to the assumption that CRF might be involved in nervous regulations other than those related to the corticotropic function.

[Repercussions of anterior hypothalamic lesions on nycthemeral variations of plasma thyroxine and corticosterone in the pigeon]. / Répercussions de lésions hypothalamiques antérieures sur les variations nycthémérales de la thyroxine et de la corticostérone plasmatiques chez le pigeon.

Plasma thyroxine and corticosterone levels were determined by competitive protein binding assay, at 3 hr intervals, throughout the photoperiod. Pigeons were kept in controlled environment (21 +/- 1 degree C; 14L6-20: 10D). Intact controls exhibited low thyroxine (T4) and corticosterone (B) levels for the light phase of the photoperiod. Values were rising during the night, up to a peak at 03 hr. Electrolytic lesions were placed bilaterally in either the nucleus anterior medialis hypothalami or the n. preopticus, or the n. supraopticus. Circadian rhythms of both T4 and B were markedly altered in all lesioned pigeons, with a shift of very high T4 values to the morning times and a complete disorganization of B patterns, with very heterogeneous values. The possibility is raised that anterior hypothalamic formations participate in the endogenous oscillator circuitry in birds.

Influence of dorsal hippocampus stimulation on the excitability of medial hypothalamic neurons in the rat.

Extracellular recordings from medial hypothalamic neurons in pentobarbital-anesthetized male Sprague-Dawley rats were evaluated during electrical stimulation in the ipsilateral and contralateral dorsal hippocampus/fimbria region. Similar short latency orthodromic (excitatory or inhibitory) responses were noted from both ipsilateral and contralateral stimulation sites indicating a bilateral projection. 14.5% of neurons responded to both stimuli, usually with a similar pattern of response. When neurons were also tested with ipsilateral basal or corticomedial amygdala stimulation, 21.5-22.8% of cells displayed orthodromic responses to both the hippocampus and the amygdala stimulus. 12.5% of medial hypothalamic tuberoinfundibular neurons also responded orthodromically to the hippocampal stimulus; a preceding stimulus to the amygdala could block this response in two of three tuberoinfundibular cells. These observations provide preliminary electrophysiological analysis of the functional connectivity of hippocampal and amygdala afferents to medial hypothalamic neurons that may underlie the influence of these extrahypothalamic regions on adenohypophyseal secretions.

Hypothalamic multiunit activity correlates of the adrenocortical response to stress.

The corticotropic response to stress was studied by means of multiple unit activity (MUA) recording from the adrenocorticotropic region of the hypothalamus, and plasma corticosterone (B) determination. MUA was permanently obtained and B was measured at 2, then 5 and 10 min intervals before and after neurogenic (electrical shocks) or systemic (ether inhalation) stress was applied. Experiments were made on steady unanesthetized, unrestrained thalamic pigeons. Post-stress alterations of MUA and B were closely in parallel, exhibiting a rapid and sustained increase in firing rate and shifting by 5-10 min, in corticosteronemia. Three successive and progressively decreasing peaks of MUA and B could be observed. Basal resting values were restored by approximately 90 min. Adrenocorticotropic responses to stress appear to be modulated through neural thalamic and/or rhombencephalic mechanisms.

[Response of the hypothalamo-corticotropic system to metyrapone in pigeons]. / Etude de la réponse du système hypothalamo-corticotrope à la métopirone chez le pigeon.

Chronic catheterization and miniature recording device allowed plasma corticosterone (B) and hypothalamic multiunit activity (MUA) to be simultaneously obtained from freely behaving, awake pigeons, before and for 4 hrs after intravenous injection of metyrapone. Injection of vehicle (tartric acid : 100 mg/4 ml/kg) led to MUA and B profiles quite similar to stress-induced responses, i.e., a rapid and sustained rebounding increase in hypothalamic firing rate and, shifted by 5-10 min, in plasma B. These responses were progressively attenuating for 90-120 mn. Metyrapone administration induced first a rapid and short MUA and B increase. Then both parameters drastically decreased near zero for about 2 hrs and were slowly restored to initial values within 3-4 hrs. It is suggested that metyrapone treatment inhibited both peripheral (B synthesis) and central (hypothalamic neurons) levels of the corticotropic axis.

Adrenocrotical responses to systemic or neurogenic stress and to hypothalamic stimulation in chronically catheterized thalamic pigeons.

Thalamic and intact pigeons were equipped with a chronic arterial catheter and with a miniature electronic device for hypothalamic telestimulation. Chronic catheterization allowed for repetitive blood sampling in freely moving birds subjected to either systemic (ether inhalation) or neurogenic (electrical foot shocks) stress and to electrical stimulation of the hypothalamic corticotropic area. Corticosterone levels were determined by protein binding assay at 2-, then 5- and 10-min intervals, for 100 min. Basal and experimentally modified plasma corticosterone concentrations were not different in thalamic and intact pigeons. Corticosterone profile exhibited episodic increase including three peaks at 12, 35 and 60 min after stress application. Only the first peak of plasma corticosterone appeared after hypothalamic stimulation. It is suggested that extrahypothalamic neuronal networks are responsible for the long-lasting repetitive adrenocorticotropic response to stress, which are not involved in the single response to hypothalamic stimulation itself. Furthermore, such extrahypothalamic neuronal networks shoudl be located at the diencephalic or rhombencephalic level since hemispherectomized pigeons exhibited the same profile of stress-induced episodic hypercorticosteronemia as seen in intact birds.

Does the nucleus raphes participate in the regulation of resting and stress-induced hypothalamic-pituitary-adrenocortical activity in the pigeon?

Extensive multiple electrolytic lesions were placed into the nucleus raphes of the brain stem in the pigeon. Diurnal pituitary-adrenocortical rhythmicity appeared not to be altered and basal plasma corticosterone level remained quite normal in raphe lesioned birds. Electrical stimulations through permanently implanted electrode were delivered in various central nervous structures in unanaesthetized, freely moving pigeons. Stimulations of nucleus raphes and of various parts of formatio reticularis led to a significant rise in plasma corticosterone within 16 to 19 min after the beginning of the stimulating session. Then, plasma B came again to initial level within 15 minutes. Stimulations of the corticotropic area of the hypothalamus (n. posterior medialis hypothalami) and of archistriatum dorsalis induced an early plasma corticosterone increase occurring immediately after the stimulating burst (10 min). Stimulating the n. septum medialis also had an immediate, but reverse (decrease) effect on plasma corticosterone level. Stress-induced pituitary-adrenal cortical activation exhibited a temporal pattern quite similar to that observed after brain stem (n. raphes or formatio reticularis) stimulation. It is suggested that these various limbic and brain stem areas might be involved in some "limbic system-midbrain circuit" with two components : The forebrain component might be involved in the regulation and diurnal modulation of basal hypothalamic-pituitary-adrenocortical function, the brain-stem component interferring with stress-induced responses.

Comparison between hypothalamic multiple-unit activity and corticotropic function after bilateral destruction of the hippocampus.

Multiple unit activity (MUA) was recorded from the adrenocorticotropic area (n. posterior medialis hypothalami, PMH) of unrestrained resting pigeons throughout the 24 h period and compared with plasma corticosterone levels (B). Bilateral electrolytic lesions of the hippocampus suppressed diurnal variations of MUA and B. Both parameters were stabilized at a steady high level whereas complete neural isolation of the basal hypothalamus led to stabilized intermediate plasma B level and MUA pattern.

Retrograde transport of horseradish peroxidase from the preoptic-anterior hypothalamic region to retinal ganglion cells in quail.

Horseradish peroxidase was injected in the preoptic-anterior hypothalamic area of intact male quail and reacted with 3,3'-diaminobenzidine and hydrogen peroxide in eye sections. Cells labelled with peroxidase reaction product were found in the ganglion layer of the retina. One possible function of such retinal-hypothalamic connections might be to participate in the photosexual reflex.

Multiple unit activity recording in the corticotropic area of the deafferented hypothalamus and corticosteronemia in the pigeon.

10 Diurnal variations in both multiple unit activity and plasma corticosterone level were suppressed after complete neural isolation of the basal hypothalamus in the pigeon. 20 It is suggested that the circadian activity of the hypothalamic pituitary corticotropic unit partially depends upon the inhibiting influence from the hippocampic-septal structures.

Comparison between hypothalamic, hippocampal and septal multiple unit activity and basal corticotropic function in unrestrained, unanesthetized resting pigeons.

Multiple unit activity (MUA) was obtained from various forebrain regions in unanesthetized, unrestrained resting pigeons throughout the whole photoperiod and compared with plasma corticosterone levels. The pattern of electrical activity recorded from the adrenocorticotropic area of the hypothalamus showed diurnal variations which paralleled the plasma corticosterone fluctuations during the 24 h photoperiod. Both parameters were low in the late afternoon and the evening and high in the early morning. Hypothalamic activation slightly preceded the peak o corticosteronemia. Conversely, in hippocampal (H) and septal (S) regions, the peak of MUA occurred in phase opposition with respect to the hypothalamic peak, and there was a marked decrease of firing rates at the moment when adrenocorticotropic activation was initiated.