H. Moor

Fine structure in freeze-etched allium cepa L. root tips

Allium cepa root tips were freeze-etched with results indicating that the technique can be applied advantageously to ultrastructural studies in the multicellular tissues of higher plants. The picture of cell and membrane structure which emerges, confirms in most respects the picture observed after chemical fixation and positive staining. In addition, freeze-etching permitted determination of pore size and revealed surface structures not seen in normally sectioned preparations.

The influence of high pressure freezing on mammalian nerve tissue

SummaryVitrification of biological specimens in liquid nitrogen can be achieved under high pressure (2,100 bars). This procedure obviates the use of aldehyde fixation and cryoprotection (glycerol). The present work demonstrates its applicability to the freeze-etching of mammalian brain tissue. Freeze-fracture replicas from rat cerebellar cortex and subfornical organ prepared by this method are compared to conventionally processed material using aldehyde fixation, glycerination and freezing with Freon. The formation of large ice crystals is prevented in tissue blocks up to 0.5 mm thick; deep etching is markedly enhanced. Cytoplasmic microstructures such as mitochondrial cristae, microtubules and microfilaments, are readily observable against a finely granulated cytosol matrix. An additional advantage is the combined application with freeze-substitution.

The fine structure of the perineural endothelium

SummaryFine strands of motor nerves were examined with the electron microscope using thin section as well as freeze-etching techniques. The specimens were taken from frog cutaneous pectoris nerve, rat sciatic nerve, mouse and shrew phrenic nerves and from human skin nerves. The perineural sheath (Henle, Ranvier, Key and Retzius) consists of one to several concentric laminae of endothelial cells; it encases nerve fascicles and eventually individual nerve fibers and terminals. The endothelial cells are extremely thin and fitted together smoothly by overlap and dove-tailing of their border zones. The cell contacts are formed by continuous zonulae occludentes, often reinforced by maculae adhaerentes, and in depth they comprise 3–15 strands with an average of 5–6 strands per junction. The membranes of endothelial cells are studded with attachment sites and stomata of plasmalemmal vesicles suggesting a high level of pinocytotic activity. This phenomenon is by no means restricted to the external laminae of the endothelial sheath. Each endothelial lamina is vested with basement membranes on both (epineural and endoneural) sides, and the spaces between laminae contain a few collagen fibers and fibroblasts. Occasionally, punctate tight junctions are seen between laminae. Cytological evidence supports the hypothesis that the perineural endothelium provides a relatively tight and highly selective barrier separating the peripheral nerves from surrounding tissue and its extracellular fluid spaces. This effect is achieved on the one hand by the sealing of pericellular spaces and on the other hand by a membrane controlled transcellular transport mechanism (pinocytosis), both of which are enhanced by their serial arrangement.

Structure and ultrastructure of the frog motor endplate

SummaryThe frog motor endplate in its simplest form consists of an elongated, slender nerve ending embedded in a gutter-like depression of the sarcolemma. This nerve terminal contains the usual synaptic organelles. It is covered by a thin coating of Schwann cell cytoplasm which embraces the terminal with thin finger-like processes from both sides, thereby sub-dividing it into 300–1000 regularly spaced compartments. The individual synaptic compartments correspond to the strings of varicosities or grape-like configurations of motor nerve terminals in endplates of other species and in the cerebral neuropil of vertebrates.Each compartment contains one or more bar-like densities of the presynaptic membrane, ‘active zones’, which are associated with the attachment sites between synaptic vesicles and plasmalemma. Active zones have a regular transverse arrangement and occur at specific loci opposite the junctional folds. The attachment sites for synaptic vesicles are at the edges of the bars which are bilaterally delineated by a double row of 10 nm particles attached to the A-face. The structural appearance of vesicle attachment sites in freeze-etch replicas corresponds to that of micropinocytosis. The active zones are often fragmented and the frequency of their association with vesicle attachment sites is highly variable.The junctional folds are characterized by “specific sites” in which intramembranous particle aggregations occur at relatively high packing density (7500/μm2). These sites are located opposite the active zones at the juxtaneural lips, a location where one would expect ACh-sensitive receptors on the postsynaptic membrane.

Platin-Kohle-Abdruck-Technik angewandt auf den Feinbau der Milchröhren

Das Auflosungsvermogen von vorbeschatteten Abdrucken wird durch die Schichtdicke, die Eigenstruktur und die Dichte des Beschattungsmaterials bestimmt. Die Eigenschaften des kontrastreichen, schwerloslichen Platins als Beschattungsmaterial konnen durch Beimischung von Chrom oder Kohle bis zu einem gewissen Grade verbessert werden. Die praktisch erzielten Auflosungen von 30–40 A entsprechen den theoretisch zu erwartenden. Durch indirekte Aufdampfung der Kohle ist es moglich, dunnste Tragerfilme von genugender Tragfahigkeit herzustellen; ihre Schichtdicke wurde mit verschiedenen, voneinander unabhangigen Methoden bestimmt. Die Entwicklung der Milchrohren von Euphorbia splendens erfolgt durch interzellulares Schlauchwachstum. Die Zellwand entsteht allein durch Apposition als Multi-net-growth. Sie ist analog den Verhaltnissen in Faserzellen und Tracheiden in eine Primarwand, Ubergangslamelle und Sekundarwand gegliedert; eine Tertiarlamelle fehlt. Die Primarwand weist Folientextur auf, die Ubergangslamelle ein in zwei Richtungen uberkreuzt verwobenes Fibrillen-system, die Sekundarwand einen 50- bis 100-schichtigen Lamellenkomplex mit Schraubentextur, in welchen der Umlaufsinn der Schraube von einer Schicht zur andern wechselt. Die Gleichzeitigkeit von Flachen- und Dickenwachstum wird einerseits durch die in der Folge der Lamellen netzartig erscheinende Textur der Zellulose und anderseits durch den hohen Gehalt an leicht hydrolysierbarem Pektin ermoglicht.

Specific arrangements of membrane particles at sites of exo-endocytosis in the freeze-etched neurohypophysis

SummaryImages have been obtained from freeze-etch replicas of neurohypophyses which are consistent with the view that orderly arranged aggregates of membrane particles occur in regions where fragments of membrane are being added to and taken away from the plasma membrane during secretion. Aggregates of particles included rosette-like and necklace-like patterns similar to those described by other authors at sites of exocytosis and endocytosis.

Synaptic vesicles in electron micrographs of freeze-etched nerve terminals.

Freeze-etched neuropil of the cat subfornical organ was examined with the electron microscope for synaptic vesicles. Round vesicles were found exclusively in both unfixed and aldehyde-fixed specimens. Range of diameters and histograms failed to differ significantly between freeze-etched and conventionally prepared material. The mnode of distribution of diameters was approximately 500 angstroms. Round stomata (approximately 350 angstromns in diameter) were found at the outer surface of the plasmalemma of nerve terminials; they are interpreted as pinocytotic vesicles.

Solitary membrane associated particles in normal and experimentally enlarged synaptic vesicles

Freeze-etch replicas of synaptic vesicles were obtained from the vertebrate central nervous system and from the frog motor endplate. Large solitary particles were found to be associated mainly to the cytoplasmic fracture face of the vesicle membrane. They are especially conspicuous in the initial stages of Wallerian degeneration when the synaptic vesicles are abnormally large. The finding of these particles bears a striking resemblance to the previous observations of cytochemically verified vesicular calcium binding sites. The possible role of these particles for control by Ca(2+) of transmitter release is discussed.