Walther Hild

Das morphologische, kinetische und endokrinologische Verhalten von hypothalamischem und neurohypophysärem Gewebe in vitro

Zusammenfassung1.Die im Hinterlappen der Hypophyse vorkommenden Gliazellen sind mit den protoplasmatischen Astrocyten anderer Hirngebiete identisch. Es ist nicht gerechtfertigt, ihnen morphologisch oder funktionell unter der Bezeichnung „Pituicyten“ eine Sonderstellung einzuräumen, zumal sich keinerlei Anhaltspunkte dafür bieten, daß die Zellen sekretorisch tätig sind. Im lebenden Zustand bilden die protöplasmatischen Astrocyten des Hinterlappens und anderer Hirngebiete dreidimensionale Netzwerke, in denen mit histologischen Methoden keine Zellgrenzen festzustellen sind. Nach längerem Aufenthalt in vitro können sich jedoch einzelne Zellen aus diesem Netzverband ablösen und epithelartige Form annehmen, was als ein Anpassungsvorgang an die neuen Umgebungsbedingungen und auf das Fehlen von Nervenfasern zurückgeführt wird, die in vivo für die Form der Gliazellen von großer Bedeutung sein dürften. Mit Hilfe von kinematographischen Zeitrafferaufnahmen werden an den protoplasmatischen Astrocyten charakteristische schüttelnde Bewegungen gezeigt, die von den Bewegungen der Oligodendrocyten unter den gleichen Bedingungen (Pomerat 1951) unterschiedlich sind.2.Eine Hormonbildung findet in den Explantaten von Hinterlappengewebe nicht statt. Die mit dem Speicherorgan in die Kulturen übertragenen Hormone werden im Verlauf der ersten 10 Tage in vitro völlig zerstört. Wird hormonfreies Hinterlappengewebe ausgepflanzt, so lassen sich in den Kulturen zu keiner Zeit Hormone nachweisen. Das Vorhandensein oder Nichtvorhandensein von Hormonen geht parallel mit dem Verhalten der histologisch darstellbaren Trägersubstanz.3.Ganglienzellen der Nuclei supraoptici und paraventriculares von erwachsenen Tieren lassen sich ohne größere Form- und Strukturveränderungen in vitro für längere Zeit erhalten. Nisslschollen sind in lebenden Nervenzellen einwandfrei zu beobachten. Typische Neurofibrillen treten jedoch erst nach Silberimprägnation in Erscheinung. Eine im Leben sichtbare feine Streifung des Cytoplasmas könnte mit dem endoplasmatischen Reticulum Palades und Porters (1952) identisch sein (vgl. Palay und Wissig 1953). Der Nucleolus führt im lebenden Ganglienzellkern deutliche Bewegungen aus. Die Zellkernmembran tritt in lebenden Nervenzellen nicht deutlich in Erscheinung, sondern wird erst unter der Einwirkung von Fixantien oder beim Zelltod aus anderen Gründen scharf ausgezogen. An den Ganglienzellfortsätzen werden mit Hilfe des Zeitrafferfilms Cytoplasmaströmungen in distaler Richtung festgestellt, die nach Erhöhung des osmotischen Drucks des Kulturmediums beschleunigt ablaufen.Sekretorische Erscheinungen an den Nervenzellen in vitro wurden nur in äußerst geringem Grade beobachtet, was auf den Mangel an physiologischem Anreiz zur Sekretbildung zurückgeführt wird. Einzelne Stadien des Ablaufs von Ganglienzelldegeneration infolge starker Lichteinwirkung und Sauerstoffmangel werden mit Hilfe der Filmaufnahme verfolgt. Die Nervenzellen zeigen sich als die trägsten Elemente in der Gewebekultur. Aktive Ortsveränderungen wurden nie beobachtet, jedoch können Ganglienzellen offenbar über kleinere Strecken von anderen wandernden Zellen passiv mitbewegt werden. Mitosen in Ganglienzellen wurden nicht beobachtet.4.Die Fähigkeit der Fortsatzregeneration an neurosekretorischen Ganglienzellen des erwachsenen Organismus in vitro ist groß, tritt jedoch erst relativ spät nach Auspflanzung in Erscheinung. Der längste gemessene Fortsatz hatte eine Länge von 7,78 mm bis zu einer Stelle, wo er sich in dichterem Zellgewebe verlor. Etwaige Gründe für den gewundenen Verlauf der Nervenfasern in vitro werden diskutiert.5.Die an den Fortsätzen sekretorischer Ganglienzellen in Schnittpräparaten sichtbaren varikösen Anschwellungen und Herringkörper lassen sich in versilberten Kulturen ebenfalls nachweisen. Innerhalb dieser Gebilde wird eine Entbündelung der im Axon dicht zusammenliegenden Neurofibrillen beobachtet.6.Geschädigte in vitro befindliche Ganglienzellformen besitzen keine Nisslschollen und Neurofibrillen. Ihr körniges Cytoplasma zeigt eine starke Affinität zu sauren Farbstoffen und Silbersalzen.7.Nach vitaler Methylenblaufärbung werden zahlreiche Nervenzellen in direktem cytoplasmatischem Zusammenhang mit anderen Ganglienzellen gefunden.

Myelogenesis in cultures of mammalian central nervous tissue

Summary1.Myelin formation around newly grown axis cylinders of Purkinje cells from the cerebellum of kittens has been observed in vitro. Myelogenesis appears to originate from cuffs or collar-like structures at peculiar sites along the course of the axons from which it spreads out in both directions.2.Oligodendrocytes or astrocytes could not be shown to be involved in this process as far as could be determined with the aid of the light microscope.3.A working hypothesis is introduced suggesting that myelin in cultures of the central nervous system is produced directly by the axoplasm. How far glial cells contribute indirectly to this process requires further study.

Histotypic organization of the rat retina in vitro

SummaryRetinae from two- and three-day-old rats were explanted in plasma clots and grown in vitro with the flying coverslip method. After seven to seventeen days in culture, the retinal tissue was fixed with aldehydes and osmium tetroxide and embedded for examination with the electron microscope. Study of cross sections (perpendicular to the coverslip) revealed a histotypic pattern of organization, especially in the thicker regions of the explants. Layering of cells quite similar to that in the intact retina was seen to develop from the relatively primitive, explanted retinal epithelium. However, each layer contained fewer cells than its counterpart in vivo. All major neuronal cell types were distinguished by their location and cytological characteristics. Development of the saccules of sensory cell outer segments was observed to occur in vitro by an infolding of the plasma membrane. Synaptic ribbon complexes developed to the mature form in the outer plexiform layers, while conventional synapses were numerous in the inner plexiform layers. Synaptic ribbons were also seen in bipolar cell axons in the inner plexiform layers. Amacrine and ganglion cells in these regions were relatively sparse. A survey of posterior regions of noncultured three-day-old rat retinae showed no synapses of any sort; therefore the synapses in the cultures formed in vitro. The retina is recommended for studies of synaptogenesis in tissue culture, for it offers several advantages over expiants from other areas of the neuraxis, including a clear layering pattern, many identifiable cell processes with characteristic synaptic relationships between them, and a well-defined sequence of developmental events.ZusammenfassungNetzhäute von 2–3 Tage alten Ratten wurden in Plasma auf Deckgläsern in Rollerröhrchen zur Kultur angesetzt. Nach 7–17 Tagen in vitro wurden die Kulturen mit Aldehyden und Osmiumsäure fixiert und für elektronenmikroskopische Untersuchung weiterverarbeitet. Gewebsquerschnitte (senkrecht zum Deckglas) zeigten histotypische Organisation, besonders in den dickeren Abschnitten der Explantate. Die Schichtung der Zellen entwickelte sich ganz ähnlich derjenigen in der Retina in situ aus dem relativ primitiven ausgepflanzten Netzhautepithel, jedoch enthielten die verschiedenen Schichten weniger Zellen als in der Retina in vivo. Alle Hauptnervenzelltypen konnten auf Grund ihrer Lokalisation und ihrer cytologischen Merkmale unterschieden werden. Die Entstehung von membranösen Lamellen in den Außengliedern der Sinneszellen konnte als Einfaltung der Plasmamembran beobachtet werden. Synaptische Bandkomplexe in ausgereifter Form wurden in der äußeren plexiformen Schicht nachgewiesen, während konventionelle Synapsen in der inneren plexiformen Schicht häufig angetroffen wurden. Synaptische Bänder waren ebenfalls in den Axonen bipolarer Zellen in der inneren plexiformen Schicht nachweisbar. Amakrine und Ganglienzellen waren in diesen Regionen ziemlich selten vertreten. Da die Untersuchung von nicht kultivierten Netzhäuten drei Tage alter Tiere keinerlei Synapsen zeigte, wird geschlossen, daß die Synapsen in den Kulturen in vitro entstanden sein müssen. Die Netzhaut stellt ein günstiges Modell für die Synaptogenese in vitro dar, indem sie verschiedene Vorzüge vor Explantaten aus anderen Regionen des Zentralnervensystems aufweist, nämlich eine klare Schichtung, zahlreiche identifizierbare Zellfortsätze mit charakteristischen synaptischen Beziehungen und eine wohl definierte Folge von Entwicklungsvorgängen.

Cell types and neuronal connections in cultures of mammalian central nervous tissue

SummaryVarious neuronal cell types surviving in cultures of cerebellum, brain stem and cerebral cortex of new-born rats and kittens were described. The regenerative power of neurons in these explants was expressed by the growth of new processes that reached a length of several millimeters. However, nerve fiber regeneration and growth followed an entirely erratic pattern as evidenced by sometimes extensive fiber convolutions.Feed-back collaterals of axonic processes that returned fibers to their own cell bodies or their dendrites were a common feature in many neurons. Short chains of interconnected neurons were detected in a number of cultures. The connections between these neurons were established by apposition of fine axon terminals to presumable postsynaptic cell bodies or dendrites but not by true synaptic boutons. In numerous instances, synapses of the bouton type that appeared morphologically normal could be shown to represent remnants of severed connections since the presynaptic fibers proved to be isolated fiber fragments. Under the sterile conditions of the culture environment these structures apparently persisted without considerable morphological alteration. Evidently, most neurons in our cultures were reduced to the status of isolated cells due to extensive de-afferentiation at the time of explantation. Moreover, there was no evidence for the establishment of new synaptic connections between regenerating neurons.

Neuroglia Electrically Coupled to Neurons

Applied electric current is transmitted between mammalian glial cells grown in tissue culture. A similar electrical coupling exists between certain neurons as well as between neuroglia and neurons. Although this phenomenon may be a peculiarity of mammalian neural cells maintained in culture, it may, on the other hand, represent a phenomenon with greater neurophysiological significance, a process whereby neurons can become silent.

Mitochondrial alterations in cultures of the central nervous system as observed with the electron microscope

Summary and conclusionElectron microscope observations on sections prepared from cultures of the cerebellum and the region of the mesencephalic nucleus of the trigeminal nerve of young kittens reveal a profusion of complex bodies that can be identified as lipid inclusions. These inclusions are not limited to any type of cell within the culture and can be found in newly formed myelinated fibers as well as cells and parts of cells that represent original explant material. Since the lipid droplets, except the very largest, have the sizes and distribution of mitochondria, and because bodies with intermediate features are numerous, the lipid droplets are considered to be transformed mitochondria.

Ependymal cells in tissue culture

SummaryCiliated ependymal cells from various locations of the ventricular system of mammals showed outgrowth either in the form of epithelial sheets or in the form of “pools” and elongated double rows, dependant on the site from which the ependyma was taken and on the original orientation of the cells in respect to the cover slip.The ependymal cilia in such cultures were shown to be moving at a rate of 51/2 to 6 times per second. The movement of the cilia of adjacent cells was apparently well coordinated causing the surrounding fluid to flow in a particular direction.The effect of „physiological“ saline, morphine sulfate, ethyl and methyl alcohol on the ciliary motility has been studied in living cells with phase contrast cinematography.

Electron microscopic observations of synaptic endings in cultures of mammalian central nervous tissue

SummarySynapses were found in rat cerebellar and brainstem cultures with the electron microscope. Three distinct types of synaptic terminals were described. The similarity between synapses found in vitro and in vivo was emphasized.

Observations on neurons and neuroglia from the area of the mesencephalic fifth nucleus of the cat in vitro

SummaryNeurons from the mesencephalic root of the fifth nucleus from the kitten have been maintained in vitro for periods up to 70 days. During this time they retained their characteristic form and structure as seen in sectioned material. Axonic processes as long as 8 mm have been measured. The occurrence of recurrent axons intertwining with dendritic arborizations of the same neuron is discussed. Modulations are described for astrocytes in relation to their possible dependence on the scarcity of connective tissue elements and a lack of an outer mesenchymal tissue zone in cultures of mesencephalic explants.

The behavior of retinal tissue in vitro, light and electron microscopic observations

SummaryRetinae from two day old rats were used in this study and the cultures were handled according to standard methods used in this laboratory. In the first few days of cultivation an abundant outgrowth of nerve fibers into the cell-free medium was observed. These fibers later degenerated and by the beginning of the second week they had completely disappeared. In the living cultures, differentiating ganglion cells, bipolar and horizontal neurons could be seen in the main explant in association with various types of glial cells. Rod cells became arranged as epithelial sheets or as clusters of cells which often formed rosettes. The nuclei of these sensory cells possessed a characteristic chromatin pattern by which they always could be differentiated from other cells in the cultures. Cytoplasmic extensions that developed from the free surface of the sensory rod cells were observed within a week following explantation. A “limiting membrane” separated these extensions from the nucleated part of the rod cells. Morphologic details of the different neuronal cell types could be demonstrated in cultures by Bodians silver impregnation technique.With the electron microscope, retinal development in culture was observed and compared to the development of the retina of the intact eye. Cilia developed from processes extending from the rod cell free surface. These processes were the rod cell inner segments in which many mitochondria were seen. At the bases of these segments terminal bars developed forming the “outer limiting membrane”. In the area of the terminal bars microvillous extensions projected between the rod cell inner segments. After twelve days in vitro a bulb-like enlargement containing a lamellar membrane system developed at the end of the cilium. This bulb-like enlargement was a beginning of the rod cell outer segment. The lamellar system did not acquire the symmetry or precise organization during cultivation that was observed in the retina of the intact eye. The distinguishing characteristics of individual neuronal cell types seen in cultivated retinae were the same as those described for their counterparts in the retina in situ, but regular plexiform layers failed to develop. Likewise, there were no indications of typical synapses in the neuropils of the cultures. There were many processes containing vesicles similar to those in presynaptic endings and mitochondria but membrane thickenings were not apparent.The results indicate that the retina cultivated in vitro does not behave as an organized entity. The component cells dissociated more and more with time, and developmental differentiation was observed only at the cellular level.