Distribution of arrestin-like protein and beta-subunit of GTP-binding proteins in quail choroid plexuses.

Monoclonal antibodies (Mabs) directed against retinal arrestin (S-antigen) were used to detect and characterize this protein in choroid plexus (CP) of quails maintained during eight days, either under long-day photoperiods or in constant darkness. Immunocytochemistry and Western blotting confirmed the presence and the distribution of an arrestin-like protein in quail CP. Arrestin-like immunoreactivities in CP were compared with those obtained with Mabs to beta 36-subunit of G proteins (G beta), alpha-subunit of transducin and rhodopsin. Rhodopsin-like and transducin-like proteins could not be detected in choroidal cells, whereas intense positive reactions were observed with anti-G beta and anti-arrestin Mabs. The strongest immunoreactivities were found in choroidal ependymocytes of the lateral and IIIrd ventricles. In CP epithelial cells lining the IVth ventricle, very weak or no immunoreactivity could be detected with Mabs to arrestin, while Mab against G beta subunit always provided a positive reaction. In quails maintained in constant darkness, arrestin- and G beta-immunoreactivities of CP epithelial cells displayed changes in cellular distribution and intensity (decrease or disappearance of the immunoreactions). The strong arrestin-like immunoreaction located in the apical region of ependymocytes suggests the preferential association of the protein with choroidal microvilli and a possible role in cerebrospinal fluid production assumed by CP cells.

Effects of hypothalamic deafferentation on basal and stress-induced adrenocortical acitivity in the pigeon.

Partial and complete deafferentation of the hypothalamus of the pigeon was performed using a modified Halasz-Pupp microknife. After complete neural isolation the plasma corticosterone level stabilized at a point intermediate between the morning and evening levels found in intact pigeons. No diurnal rhythm was observed and the response to neurogenic stimulus (restraint) was suppressed. Ether stress, however, induced a rise in plasma corticosterone. Posterior deafferentation had no effect on the diurnal corticosterone rhythm but did block the rise normally found after restraint. Anterior deafferentation did not suppress the stress-induced response but provided the cuts were large enough they inhibited the diurnal corticosterone rhythm. It is suggested therefore that the neural afferents to the hypothalamus which are necessary for diurnal fluctuations in pituitary-adrenal function pass through a sector located anteriorly between 45 degrees and 60 degrees on either side of the mod-line while stress-induced adrenocortical activation is triggered through posterior connexions.

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.

Choroidal readaptation to gravity in rats after spaceflight and head-down tilt.

To determine when choroidal structures were restored after readaptation to Earth gravity or orthostatic position, fine structure and protein distribution were studied in rat choroid plexus dissected either 6 h [Space Life Sciences-2 (SLS-2) experiments] or 2 days [National Institutes of Health-Rodent 1 (NIH-R1) experiments] after a spaceflight, or 6 h after head-down tilt (HDT) experiments. Apical alterations were noted in choroidal cells from SLS-2 and HDT animals, confirming that weightlessness impaired choroidal structures and functions. However, the presence of small apical microvilli and kinocilia and the absence of vesicle accumulations showed that the apical organization began to be restored rapidly after landing. Very enlarged apical microvilli appeared after 2 days on Earth, suggesting increased choroidal activity. However, as distributions of ezrin and carbonic anhydrase II remained altered in both flight and suspended animals after readaptation to Earth gravity, it was concluded that choroidal structures and functions were not completely restored, even after 2 days in Earth's gravity.

Heart and plasma atrial natriuretic peptide (ANP) in response to long-term endurance training in rats.

Long-term endurance training effects on heart and plasma ANP were investigated in male Wistar rats. Maximal O2 uptake (VO2max) was significantly higher in trained groups, when they are used as their own control. After 3, 4, and 5 weeks of endurance training, VO2max was respectively increased by 7.7% (p less than 0.05), 13.7% (p less than 0.01), and 18.4% (p less than 0.001). Plasma ANP and glomerular ANP receptor density showed no clear variations in trained rats. However, cardiac ANP content decreased significantly in left and right atrial tissues by 35-36% (p less than 0.05) after 5 weeks of training. ANP immunoreactivity was investigated to show the distribution of ANP within the atria. ANP was found in diffuse and granular forms. The diffuse pattern (immature ANP) disappeared in cardiocytes of trained rats, while the granular form persisted, especially in the left atrial tissue. These data suggest that chronic endurance training might cause a decrease in ANP synthesis with no change in ANP storage. Such results are in agreement with the hypothesis that the left atrium could be especially involved in long-term fluid volume control.

Changes in apical organization of choroidal cells in rats adapted to spaceflight or head-down tilt.

Structural changes observed in choroid plexuses from rats dissected aboard a space shuttle, on day 13 of an orbital flight (NASA STS-58 mission, SLS-2 Experiments) demonstrated that choroidal epithelial cells display a modified organization in a microgravitational environment. Results were compared with ultrastructural observations of choroid plexus from rats maintained under anti-orthostatic restraint (head-down tilt) for 14 days. In both experiment types, the main alterations observed by transmission electron microscopy, at the level of choroidal epithelial cells from the third and fourth ventricles, concerned the formation and the organization of apical microvilli, whereas pseudopod-like structures appeared. Immunocytochemical distribution of ezrin, a cytoskeletal protein involved in apical cell differentiation in choroid plexus, confirmed the structural alteration of microvilli in head-down tilted rats, Kinocilia tended to disappear from the apical surface, suggesting a partial loss of cell polarization. In addition, large amounts of clear vesicles were gathered in the apical cytoplasm of choroidal epithelial cells. Disorganization of apical microvilli accumulations of apical vesicles and partial loss of cell polarity showed that long-stays in weightlessness induced alterations in the fine structure of choroid plexus, consistent with a marked reduction of cerebrospinal fluid production.

Effects of an 11-day spaceflight on the choroid plexus of developing rats.

Cellular distributions of ezrin, a cytoskeletal protein involved in apical cell differentiation in choroid plexus, and carbonic anhydrase II, which is partly involved in the cerebrospinal fluid production, were studied by immunocytochemistry, at the level of choroidal epithelial cells from the lateral, third and fourth ventricles in normal or experimental fetuses, in parallel with the ultrastructure of apical microvilli, observed by transmission electron microscopy. We compared choroid plexuses from developing normal rats (gestational day 15 to birth) with choroid plexuses from 20-day-old rat fetuses, developed for 11 days in space, aboard a space shuttle (NASA STS-66 mission, NIH-R1 experiments), from gestational day 9 to day 20. The main changes observed in fetuses developed in space were demonstrated by immunocytochemistry and concerned the distribution of ezrin and carbonic anhydrase II. Thus, in fetuses developing in space, ezrin was strongly detected in the choroidal cytoplasm and weakly associated to the membrane in the apical domain of the choroid plexus from the fourth ventricle. Such alterations suggested that choroid plexus from rat fetal brain displays a delayed maturation under a micro-gravitational environment. In contrast, intense immunoreactions to anti-carbonic anhydrase II antibodies showed that this enzyme is very abundant in rats developed in space, compared to ground control fetuses.

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.

ANP binding sites are increased in choroid plexus of SLS-1 rats after 9 days of spaceflight.

Specific alpha-rat 28-amino acid atrial natriuretic peptide [ANP(99-126)] (rANP) binding sites in choroid plexus and meningia of rats flown for 9 days on the mission STS-40 (SLS-1) carried on the space shuttle Columbia in June 1991 were analyzed after incubation of brain sections with 125I-rANP and autoradiography, using computer-assisted microdensitometric image analysis. The number of 125I-rANP binding sites (expressed by Bmax values) in the choroid plexus of lateral and third ventricles of these rats was significantly increased (x 1.5-2.5 times), as compared with that found in ground control rats. No differences in the binding affinity (deducible from Kd values) were observed at the level of these structures. The choroid plexus from the fourth ventricle of the same rats displayed no changes in the binding capacity or affinity after the spaceflight. Meningia from the rats flown in space did not demonstrate any significant modifications of the number of 125I-rANP binding sites, but displayed a significant increase in Kd values, which suggested a reduced affinity of the meningeal ANP receptors during a 9-d spaceflight. The possibility that atrial natriuretic peptide may be involved in the regulation of fluid and electrolyte fluxes in the brain, during adaptation to microgravity, through modified expression of specific high affinity receptors, mainly in choroid plexus from forebrain or in meningia, must be considered.

Choroidal responses in microgravity. (SLS-1, SLS-2 and hindlimb-suspension experiments).

Fluid and electrolyte shifts occurring during human spaceflight have been reported and investigated at the level of blood, cardiovascular and renal responses. Very few data were available concerning the cerebral fluid and electrolyte adaptation to microgravity, even in animal models. It is the reason why we developed several studies focused on the effects of spaceflight (SLS-1 and SLS-2 programs, carried on NASA STS 40 and 56 missions, which were 9- and 14-day flights, respectively), on structural and functional features of choroid plexuses, organs which secrete 70-90% of cerebrospinal fluid (CSF) and which are involved in brain homeostasis. Rats flown aboard space shuttles were sacrificed either in space (SLS-2 experiment, on flight day 13) or 4-8 hours after landing (SLS-1 and SLS-2 experiments). Quantitative autoradiography performed by microdensitometry and image analysis, showed that lateral and third ventricle choroid plexuses from rats flown for SLS-1 experiment demonstrated an increased number (about x 2) of binding sites to natriuretic peptides (which are known to be involved in mechanisms regulating CSF production). Using electron microscopy and immunocytochemistry, we studied the cellular response of choroid plexuses, which produce cerebrospinal fluid (CSF) in brain lateral, third and fourth ventricles. We demonstrated that spaceflight (SLS-2 experiment, inflight samples) induces changes in the choroidal cell structure (apical microvilli, kinocilia organization, vesicle accumulation) and protein distribution or expression (carbonic anhydrase II, water channels,...). These observations suggested a loss of choroidal cell polarity and a decrease in CSF secretion. Hindlimb-suspended rats displayed similar choroidal changes. All together, these results support the hypothesis of a modified CSF production in rats during long-term (9, 13 or 14 days) adaptations to microgravity.

Suppression of pineal multiunit response to flash after habenular lesion in quail.

Habenular nuclei were completely destroyed in male photostimulated and nonphotostimulated quail (Coturnix coturnix japonica). Lesion of the nuclei habenulae led to a markedly increased pineal multiunit activity (MUA) pattern in both long and short daily photoperiods compared to control values. Intact, as well as habenular lesioned birds, showed a lower level of pineal MUA under long-day rearing than under short-day rearing. The decrease in pineal firing rates found in controls under repetitive flash-light stimulations was suppressed in quail bearing habenular lesions. Data are compared to results obtained in sympathectomized and blinded birds. It is suggested that some acute photic information might reach the pineal organ through retinal receptors and habenular pathways. However chronic influence of lighting on pineal MUA, mediated by the retina, does not seem to involve habenular or cervical sympathetic pathways in quail.

Influence of castration and testosterone on pineal multi-unit activity in quail.

Pineal multiple unit electrical activity was recorded in unanaesthetized quail maintained under a short or long daily photoperiod. Both spontaneous and flash-induced multiple unit firing were recorded in intact, castrated or superior cervical ganglionectomized birds. The effects of testosterone administration were tested in each experimental group. 10 Whereas orchidectomy led to a substantial rise in pineal multi-unit activity (MUA), testosterone propionate injection resulted in a markedly decreased MUA in castrates. A smaller decrease in MUA was caused by androgen treatment in intact or ganglionectomized birds. Androgen treatment of controls and ganglionectomized quail was not so effective in reducing pineal activity as was lengthening the photoperiod from 6L-18D to 18L-6D. 20 Repetitive photic stimulations resulted in a significant suppression of pineal MUA in all intact, castrated and ganglionectomized quail, whether or not testosterone was administered.

Pineal multiunit activity in conscious quail: effects of light, blinding, ganglionectomy.

Multiunit activity (MUA) was recorded from thr pineal gland in unanesthetized male Japenese quail. The influence of light regimen on spontaneous and flash-evoked firing patterns of pineal cells was investigated. Shortening the day length from 18 to 6 h/24 h resulted in a marked increase in pineal MUA. Pineal firing rate was higher during the dark than during the light fraction of the photoperiod. Flashlight stimulation always led to a marked inhibition of the pineal MUA. Cutting the optic nerves bilaterally resulted in a marked increase in the level of MUA. None of the experimental conditions altered the elevated MUA in blinded birds. Thus, photoperiod and light stimulation appeared to be important for pineal activity; however, no direct photosended quail. Superior cervical ganglionectomy resulted in slightly increased spontaneous MUA but did not inhibit pineal responses to flashlight stimulations.

[Nycthemeral variations of thyroxine levels in the quail: effect of ambient temperature]. / Variations nycthémérales de la thyroxinémie chez la caille: influence de la température ambiante.

Plasma T4 levels were determined at 2 hr intervals in male quail reared under 18 L6-24 : 6D and 26 +/- 1 degrees C. Low thyroxinemia (4.7 -5.7 mg/ml) was observed during the light phase of the photoperiod. T4 levels were sharply increasing during the first part of the dark period, up to a peak (9.2 ng/ml) at 3 a.m., and decreased again by the last part of the night. Exposure to 4 degrees C for 30 min resulted in different effects on thyroxinemia according to the time of the day. During the light phase (i.e., low resting T4 levels), cold- induced thyroxinemia was significantly increased (50% approximately). Similar effects were observed during the last part of the night. On the contrary, no thyroid response to cold could be detected during the beginning of the dark period, when basal thyroxine concentrations were sharply increasing. Participation of feed back mechanisms in such a phenomenon is suggested.U

[Role of the infundibular complex and its neural afferents in the photosexual reflex in the quail]. / Etude du rôle joué par le complexe infundibulaire et ses afferences nerveuses dans le réflexe photosexuel chez la Caille.

The photosexual reflex was suppressed in quail after bilateral preoptic lesions. However an anterior-lateral de-afferentation of the hypothalamus of previously preoptic-lesioned quail resulted in partial photo-induced testis growth. As a working hypothesis, it is suggested that the preoptic area may control some unknown extrahypothalamic influences in order to modulate the gonadotropin controlling function of the infundibular area.

Effects of bilateral lesions of the nuclei habenulae on plasma thyroxine levels in Japanese quail.

Electrolytic bilateral lesions of th nuclei habenulae were made in male, adult photostimulated quail. Habenular destruction led to a marked decrease in the plasma thyroxine level (40%), whereas sham operated birds did not differ from controls. This result appears to be somewhat different from those obtained in mammals and the mechanisms of habenular-thyroid interrelationships are unknown.

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.

Effect of short exposure to cold on plasma thyroxine in Coturnix quail: role of the infundibular complex and its neural afferents.

Exposure of control quail to low ambient temperature (4 degrees) for a short duration (15 min) led to a rapid increase in plasma thyroxine (T4) levels. A peak appeared 40 min after the cold began and was followed by a progressive and slow decline. T4 levels were elevated in birds maintained for up to 3 hr at 4 degrees. Restraint stress could be accompanied by a slight and late decrease in thyroxine concentration, indicating that glucocorticoids could partly inhibit thyroid function. Both cold and restraint stresses elicited sustained adrenocortical activation. No thyroid response to cold appeared after complete or partial neural deafferentation of the hypothalamus, indicating that cold signals were conveyed to the thyrotropic centers from peripheral and/or deep thermoreceptors through neural posterior-lateral afferents to the hypothalamus. No thalamic relay appeared to be necessary since normal thyroidal stimulation was observed after thalamic-hypothalamic disconnection. Such a response persisted in hemispherectomized quail.

Suprachiasmatic nucleus lesions abolish photoperiod-induced changes in the testis function and GnRH immunoreactivity in the mink, a short-day breeder.

Testicular activity (testis volume and plasma testosterone) and immunoreactive GnRH hypothalamic system were examined after suprachiasmatic nucleus (SCN) lesion in the mink, a short-day breeding mammal, whose sexual activity is inhibited by day lengths exceeding 10 h. In animals maintained under a natural photoperiod, SCN destruction performed during the period of maximum sexual activity (February) was shown to have no effect on onset of the testicular inactive period which begins at the end of winter and continues through spring. On the other hand, while gonadal activity began again at the end of autumn in intact animals, minks that had undergone SCN destruction remained sexually inactive until the end of the experiment period (February). The SCN could thus be crucial to the onset of sexual activity triggered by the reduction of day length, whereas onset of sexual inactivity is a spontaneous phenomenon. This was confirmed in a second experiment demonstrating that a short photoperiod (4 L:20 D), highly gonadostimulatory in intact animals, had no effect on testicular activity after SCN destruction. An immunocytochemical study of the hypothalamic GnRH system (staining intensity and number of labeled perikarya and immunoreactive endings in the external layer of the median eminence) also showed consistent by very low rates of immunoreactivity and number of labeled perikarya and endings in operated animals.

The effect of lesions of the epithalamus on plasma aldosterone and corticosterone levels in Japanese quail.

The peripheral plasma aldosterone and corticosterone concentrations were measured by CPBA and RIA method in Japanese quails in which the subcommissural organ (SCO) or the habenula had been destroyed. Lesion of the SCO reduced the plasma aldosterone level by 50%. The lesion of n. habenularis medialis and lateralis on the other hand did not affect the plasma corticosteroid level. It is suggested that in birds the SCO takes part in a multifactorial regulation of aldosterone secretion.