Weitere enzymhistochemische und fluoreszenzmikroskopische Studien an den Plexus chorioidei und an der Paraphyse von Rana temporaria L.

Zusammenfassung Die Epithelzellen der Plexus chorioidei ventriculi III und IV und der Paraphyse von Rana temporaria L. zeigen histochemisch eine deutliche Aktivität der Phosphorylase, Glucose-6-Phosphat-Dehydrogenase und Aldolase. Die Aktivität der Uridindiphosphoglucose-Glycogen-Transferase ist im Plexusepithel der Frösche gering. Glucose-6-Phosphatase läßt sich in den Plexus chorioidei und in der Paraphyse von Rana temporaria nicht darstellen. Phosphorylase, Glucose-6-Phosphat-Dehydrogenase und Aldolase zeichnen sich durch ein charakteristisches, verschiedenartiges Verteilungsmuster aus. Der Einfluß von Adrenalininjektionen auf die Aktivität der obengenannten Enzyme des Plexus-und Paraphysenepithels und funktionelle Zusammenhänge mit der gleichzeitig eintretenden Entspeicherung ihres Glykogenvorrats werden erörtert. In fluoreszenzmikroskopischen Untersuchungen mit der Falck-Hillarp Methode läßt sich weiterhin beobachten, daß nach Adrenalingaben im Plexusepithel der Frösche ein Fluorophor auftritt, das bei Kontrolltieren vollständig fehlt. Im Plexusstroma finden sich nur vereinzelt adrenerge Nervenfasern.

---

Enzyme histochemical (carbohydrate metabolism) and fluorescence microscopic (biogenic amines) investigations of the choroid plexuses and the paraphysis in Rana temporaria L.

Summary Epithelial cells of the choroid plexuses and paraphysis in Rana temporaria L. show histochemically distinct phosphorylase, glucose-6-phosphate dehydrogenase, and aldolase activities. The uridine diphosphate glucose-glycogen transferase-reaction of the choroid epithelium is very weak, and the glucose-6-phosphatase-reaction of the choroid plexuses and the paraphysis is negative. Choroid plexuses and paraphysis differ in their distribution patterns of phosphorylase, glucose-6-phosphate dehydrogenase, and aldolase activities. Injections of epinephrine into the dorsal lymph sac increase the histochemically detectable activity of phosphorylase, glucose-6-phosphate dehydrogenase and aldolase, and deplete the glycogen stores of the choroid epithelium. After administration of epinephrine into the dorsal lymph sac, intensely fluorescent material is observed in the choroid epithelium with the Falck-Hillarp method. The adrenergic innervation of the choroid plexus of Rana temporaria is sparse.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Elektronenmikroskopische und histoautoradiographische Befunde zur Puromycinwirkung auf ausreifende Plexus chorioideus-Zellen in vitro

Zusammenfassung Der Einfluß von Puromycin auf die Inkorporierung von Leucin-³H wird an kultivierten Plexus chorioideus-Zellen 9 Tage alter Hühnerembryonen untersucht. Die Kulturen zeigen unter Einwirkung von Puromycin eine deutliche Hemmung der Aufnahme von Leucin-³H. Die Aufnahme der markierten Substanz ins Cytoplasma wird im Vergleich zu Kontrolluntersuchungen prozentual mehr gehemmt als die Aufnahme des Leucins in den Kern.Unmittelbar nach dem Zusatz von Puromycin zum Nährmedium treten in den kultivierten Plexus chorioideus-Zellen Ansammlungen von multivesicular bodies und z. T. auch zahlreiche Lysosomen auf. Beide Organellen verschwinden im Verlauf der weiteren Kultivierung wieder fast vollständig aus den Zellen.Eine neuerliche Zugabe von markiertem Leucin nach der Puromycingabe zeigt, daß die beobachteten Wirkungen reversibel sind und daß die Puromycinwirkung nicht länger anhält, als diese Substanz im Nährmedium vorhanden ist.

---

On the influence of puromycin on embryonic choroid plexus cells in vitro. An electronmicroscopic and histoautoradiographic study

Summary The influence of puromycin on the incorporation of leucin-³H into cells of choroid plexus of nine day old chicken embryos is studied.Puromycin causes a strong inhibition of the incorporation of leucin-³H into the cultures. Compared with controls, the inhibition of the uptake of the labelled substance is more pronounced in the cytoplasm than in the nucleus.Immediately after puromycin is added to the tissue culture medium an accumulation of multivesicular bodies and—to a certain degree—of lysosomes appears in the cells of the choroid plexus. Both organells disappear almost completely during the cultivation period.A second incubation with labelled leucin after puromycin shows that the observed effects are reversible and that puromycin acts only as long as it is present in the culture medium.The embryonic cells of choroid plexus develop normally even though treated with puromycin.

Die unreifen Plexus chorioideus-zellen entwickeln sich trotz der Puromycingaben normal.Das morphologische Differenzierungsergebnis wird nicht verändert.     

Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat

Summary The pelvic ganglia supply cholinergic and noradrenergic nerve pathways to many organs. Other possible transmitters are also present in these nerves, including peptides. Multiple labelling immunofluorescence techniques were used in this study of the male rat major pelvic ganglion (MPG) to examine: (1) the peptides present in noradrenergic (tyrosine hydroxylase (TH)-positive) and non-noradrenergic (putative cholinergic) neurons, and (2) the types of peptide-containing nerve fibres closely associated with these two groups of neurons. The distribution of the peptide galanin (GAL) within the MPG was also investigated. All of the TH-neurons contained neuropeptide Y (NPY), but none of the other tested peptides. However, many NPY neurons did not contain TH and may have been cholinergic. TH-negative neurons also displayed vasoactive intestinal peptide (VIP), enkephalin (ENK) or GAL. VIP and NPY formed the most common types of putative cholinergic pelvic neurons, but few cells contained both peptides. Many ENK neurons exhibited VIP, NPY or GAL. Varicose nerve terminals surrounding ganglion cells contained ENK, GAL, somatostatin (SOM) and cholecystokinin (CCK). These peptide-immunoreactive fibres were more often associated with the non-noradrenergic (putative cholinergic) than the noradrenergic neurons; two types (SOM and CCK) were preferentially associated with the non-noradrenergic NPY neurons. GAL was distributed throughout the MPG, in small neurons, scattered small, intensely fluorescent (SIF) cells, and both varicose and non-varicose nerve fibres. The nerve fibres were concentrated near the pelvic and penile nerves; most of the varicose fibres formed baskets surrounding individual GAL-negative somata.     

Transport of quinolinic acid into rabbit and rat brain

The transport metabolism of [³H]quinolinic acid in the central nervous system of rabbits and rats were studied. In vitro [³H]quinolinic acid was not readily accumulated by isolated choroid plexus. After the intraventricular injection of tracer quantities of [³H]quinolinic acid, the [³H]quinolinic acid did not enter the brain as readily as concurrently injected [¹⁴C]mannitol and was not metabolized, The permeability-surface area constant for [³H]quinolinic acid at the rat blood-brain barrier was 1.5±1.3×10^–5 sec^–1 compared to 2.8±0.4×10^–5 sec^–1 for [³H]mannitol. Our results suggest that: 1) [³H]quinolinic acid is transported in the CNS by passive diffusion and 2) is not metabolized.     

The Polarity of Choroid Plexus Epithelial Cells In Vitro Is Improved in Serum-Free Medium

Abstract: The influence of culture conditions on the development of normal characteristics of the choroid plexus epithelium has been investigated in vitro with respect to polarity, barrier properties, transport, and secretory activity. Withdrawal of serum supplement in the culture medium of cells grown on filters caused morphologically visible changes by an increased trimming of microvilli at the apical membrane side, which is accompanied by an increased expression of the Na⁺,K⁺-ATPase. Moreover cells under serum-free conditions exhibit structural changes in tight junctional zonula occludens protein-1 (ZO-1) organization, a reduced permeability, and a drastically increased electrical resistance from 150 Ω· cm² in the presence of serum to 1,500 Ω· cm² after serum withdrawal. Under these conditions, cell monolayers are able to build up a transcellular proton gradient and to secrete fluid into the upper (apical) filter compartment, which is accompanied by a polarized secretion of proteins like transthyretin. Active transport of the dyes fluorescein and phenol red by the organic anion transporter is found to be driven by the Na⁺,K⁺-ATPase. We come to the conclusion that removal of serum favors the differentiation process of the plexus epithelium in vitro, which brings the cell culture model closer to the physiological situation in vivo. We present preliminary evidence that epidermal growth factor may be one component in serum preventing the proper in vitro differentiation.     

A developmental study of the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder

A histochemical investigation of age-related changes that occur with respect to the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder was carried out. In all age groups examined (embryonic stages day 34 and 52, 2 to 4-day old, 6-month old and 2-year old), the mean percentage of NADPH-diaphorase-positive neurons per ganglion was obtained by taking the number of neurons that were immunoreactive to protein gene product 9.5 (a general neuronal marker) as 100%. In addition, the possible co-existence of NADPH-diaphorase and nitric oxide synthase in the ganglionated plexus of 2 to 4-day old and 6-month old guinea-pig gallbladder was investigated. NADPH-diaphorase was not present in the ganglionated plexus of the gallbladder at embryonic day 34. At embryonic day 52, all the protein gene product 9.5-immunoreactive neurons showed positive staining to NADPH-diaphorase; this dropped to a minimum at 2–4 days (26.7%), rose slightly at 6 months (33.6%), and finally returned close to the 100% value at 2 years. In the gallbladders of 2-year old guinea-pigs, some (3 out of 10) ganglia were devoid of protein gene product 9.5-immunoreactive neurons, but NADPH-diaphorase-stained granules were found within the ganglia. However, all those neurons that were immunopositive to protein gene product 9.5 also expressed NADPH-diaphorase. Moreover, NADPH-diaphorase-positive neurons in the gallbladder of 2 to 4-day-old and 6-month-old guinea-pigs were found to express nitric oxide synthase.     

Sodium localization in the choroid plexus

Summary The localization of sodium ion in the cat choroid plexus was studied by use of potassium pyroantimonate. The precipitates formed by the potassium pyroantimonate occur mostly on the plasma membrane in the epithelial cell and occasionally in the perivascular space. The precipitates in the epithelial cell are most numerous at the apical surface, particularly on the microvilli, and least in number at the basal and lateral surfaces. In the endothelial cell, the dense precipitates are situated on the plasma membrane as well as on the limiting membrane of the pinocytotic vesicle. Although the dense precipitates are sometimes situated on the external surface of the plasma membrane of the epithelial cell, most of them are localized on the internal surface of the plasma membrane. A similar localization of the precipitates is to be seen on the plasma membrane of the erythrocyte. When the cerebrospinal fluid/plasma ion ratio and potential gradients across the choroid plexus are considered, the precipitates on the plasma membrane would suggest a localization of sodium needed for the activation of ATPase.

---

Zusammenfassung Die Lokalisation des Natriumions im Plexus chorioideus der Katze wurde mit Hilfe von Kaliumpyroantimonat untersucht. Die durch Kaliumpyroantimonat gebildeten Niederschläge treten meistens an der Plasmamembran in den Epithelzellen und gelegentlich im perivaskulären Raum auf. In den Epithelzellen kommen die Niederschläge am zahlreichsten an der apikalen Oberfläche vor, besonders an den Mikrovilli, am geringsten an den basalen und lateralen Oberflächen. In der Endothelzelle liegen die dichten Niederschläge an der Plasmamembran und an der Grenzmembran der Pinozytosebläschen. Einige der dichten Niederschläge befinden sich an der äußeren Oberfläche der Plasmamembran der Epithelzellen, die meisten aber an der inneren Oberfläche der Plasmamembran. Eine ähnliche Lokalisation der Niedersschläge wurde an der Plasmamembran des Erythrozyten festgestellt. Wenn man das Liquor Plasma-Ionenverhältnis und die Potentialgradienten am Plexus chorioideus in Betracht zieht, liegt es nahe, die nachgewiesene Lokalisation des Natriums auf eine Aktivierung von ATPase zu beziehen.

     

Uptake and transport of immunoglobulin by the chick choroid plexus

Summary The choroid plexus from the lateral ventricles of 18-day chick embryos was cultivated as an organ in medium 199 until the degeneration of the stroma. Selected plexuses forming an empty epithelial sac were then incubated with enzyme-treated human immunoglobulin (5S-antibody) and with native human immunoglobulin (7S-antibody). Uptake of the 7S-antibody was observed after 30 min, whereas the 5S-antibody was taken up by the choroid plexus within 1 min, as demonstrated by means of the peroxidase-anti-peroxidase (PAP) technique (Sternberger 1974). The antibodies were located in conspicuous, large vacuoles of the choroid epithelium. Further experiments were performed using only 5S-antibody. In addition to the demonstration of the protein structure of this immunoglobulin, it was also shown that its binding capacity for tetanus toxoid as an antigen remains intact in the intracellular location. It was not possible to observe lysosomal degradation. Moreover, 5S-antibody was detectable in cultures first incubated with 5S-antibody for 30 min and subsequently in antibody-free medium for a further period of 7 to 11 days.The biological significance of the uptake of material from the cerebrospinal fluid and the possibility of the existence of a receptor for 5S-antibody are discussed.     

Differences in recovery processes of circadian oscillators in various tissues after sevoflurane treatment in vivo

The inhalation anesthetic sevoflurane reversibly suppresses Period2 (Per2) mRNA expression in the suprachiasmatic nucleus (SCN). However, a discrepancy exists in phase shifting of the Per2 expression rhythm between sevoflurane application in rats (in vivo application) and explants (ex vivo application). This investigation aimed to resolve this issue. First, tissues from the SCN, choroid plexus in the lateral ventricle (CP-LV), and choroid plexus in the fourth ventricle (CP–4V), which are robust circadian oscillators, and pineal gland (PG) tissue, which is a circadian influencer, were prepared from Per2::dLuc transgenic rats. Significant phase responses of bioluminescence rhythms for different preparation times were monitored in the four tissue explant types. Second, tissue explants were prepared from anesthetized rats immediately after sevoflurane treatment, and bioluminescence rhythms were compared with those from non-anesthetized rats at various preparation times. Regarding bioluminescence rhythm phases, in vivo application of sevoflurane induced phase shifts in CP-LV, CP-4V, and PG explants according to the times that rats were administered anesthesia and the explants were prepared. Phase shifts in these peripheral explants were withdrawn due to the recovery period after the anesthetic treatment, which suggests that peripheral tissues require the assistance of related tissues or organs to correct phase shifts. In contrast, no phase shifts were observed in SCN explants. These results indicated that SCN explants can independently correct bioluminescence rhythm phase. The bioluminescence intensity of explants was also decreased after in vivo sevoflurane application. The suppressive effects on SCN explants were withdrawn due to a recovery day after the anesthetic treatment. In contrast, the suppressive effects on the bioluminescence intensities of CP-LV, CP-4V, and PG explants remained at 30 days after anesthesia administration. These results suggest that anesthetic suppression is imprinted within the peripheral tissues.