C. Sandri

An electron-microscopic study of zinc iodide-osmium impregnation of neurons. I. Staining of synaptic vesicles at cholinergic junctions

Abstract The structural basis of the affinity of zinc iodide-osmium stain to nerve terminals was examined with the aid of the electron microscope at the mammalian subfornical organ and myoneural junction. It turned out that this affinity is based on the selective staining of synaptic vesicles, whose content forms an electron-opaque reaction product. The synaptic membranes, the basement membranes of junctional folds and the mitochondria remain indifferent. Certain types of synaptic vesicles gave equally negative results: (1) 800–1500Adark-cored vesicles in nerve terminals of the subfornical organ and (2) large populations of agranular spheric vesicles within spinal cord nerve terminals. These data may indicate some degree of specificity with respect to vesicular content. The possible relationship of this staining method to mechanisms of cholinergic transmission is mentioned.

Contribution to the problem of structural organization of the presynaptic area

Summary A new method of synaptic staining has been developed by using bismuthiodide block impregnation combined with uranyl acetate and lead hydroxide contrast. Synaptic junctions of the cat subfornical organ have been studied in detail with this procedure. Special emphasis was placed upon the spatial organization of presynaptic dense projections of Gray which consist of a grid with hexagonal pattern of peaks, interconnecting filaments and holes. A three-dimensional reconstruction of the presynaptic area is presented. The new impregnation spares the ‘clear’ vesicles but impregnates the large dark-cored vesicles (1000–1500A˚) rather well. The similar appearance of vesicular cores and presynaptic dense projections as well as their occasional proximity is discussed with respect to functional relationships between the two structures.

The fine structure of freeze-fractured presynaptic membranes

SummaryElectron micrographs of presynaptic membrane faces were obtained from replicas of aldehyde-fixed, freeze-fractured neuropil of mammalian and avian C.N.S. There is strong evidence that the presynaptic membrane is split and that the exposed membrane faces represent the inner and outer membrane leaflet respectively. Presynaptic sites on the inner face appear as patches adhering to postsynaptic elements and include a distinctly indented area. They bear clusters of membrane particles and a varying number of small protuberances. The latter apparently occur in presynaptic membranes exclusively and form hexagonal patterns corresponding to the ‘presynaptic vesicular grid’. Evidence is presented that the protuberances are temporary attachment sites of transmitter vesicles to the presynaptic membrane. These contacts may be identical with the sites of transmitter release.

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.

Dynamic ultrastructure of presynaptic membranes at nerve terminals in the spinal cord of rats. Anesthetized and unanesthetized preparations compared

Abstract 1. (1) Freeze-etching and thin-section electron micrographs have been used in examining the ultrastructure of presynaptic membranes in the ventral horn of the spinal cord of rats anesthetized with pentobarbital and unanesthetized. 2. (2) As seen in freeze-etched material, the main criteria useful for recognition of synaptic sites on presynaptic membranes in the anesthetized state have been confirmed in the unanesthetized state: indeed, those features are exaggerated, synaptic sites are more readily recognized and attendant ambiguities are reduced. 3. (3) The main features of synaptic site recognition are: membrane liftings, wrinklings and craters. These can all be easily recognized qualitatively in thin-sectioned and freeze-etched materials. In thin sections freeze-etched craters are seen to stand up from the presynaptic membrane like an omega profile whose neck is open and whose dome is about the size of synaptic vesicles. 4. (4) Quantitative estimates of the presence or absence of liftings, wrinklings and craters affecting presynaptic membranes show that these criteria are all significantly increased in unanesthetized as compared with anesthetized preparations. However, quantitative differences in lifting as observed in anesthetized and unanesthetized preparations could not be found in thin-sectioned material. This discrepancy may be due to simple geometric limitations affecting interpretations of thin profiles as compared with panoramic views. 5. (5) Concerning the wrinkling of the presynaptic membrane, S- and F-type synapses were not differentially affected in the anesthetized or in the unanesthetized state. 6. (6) Evidence obtained by comparing anesthetized and unanesthetized states suggests a 3-way positive correlation among behavioral states, physiological criteria for transmitter release, and presynaptic membrane activity characterized by liftings, wrinklings and craters. 7. (7) Morphological evidence here presented reinforces the vesicle hypothesis for quantal release of synaptic transmitters.

Sensorische Eingänge und synaptische Verbindungen im Zentralnervensystem von Insekten

SummaryPathways of axons from antennal receptor organs into the brain have been traced by means of anterograde experimental degeneration in Calliphora vicina and Periplaneta americana. After removal of one antennal flagellum (with the two proximal segments left intact) degenerating nervous processes were found in the glomeruli of the ipsilateral deutocerebrum. In Calliphora a great number of these axons run via a pathway dorsal to the oesophagus into the glomeruli of the contralateral deutocerebrum. A big branch of the antennal nerve of Calliphora runs toward a posterior region of the brain bypassing the glomerular region. After removal of the whole antenna including the proximal segments degenerations can be seen also in this branch on the ipsilateral side. The synapses in the glomerular region of the deutocerebrum resemble other central synapses in insects described so far (electron dense cleft, often ribbon-like dense structures within the presynaptic element near or at the synaptic membrane, clear vesicles etc.).ZusammenfassungBei Calliphora vicina und Periplaneta americana wurde mit Hilfe experimentell erzeugter anterograder Degeneration der Verlauf von Axonen antennaler Rezeptorzellen im Oberschlundganglion verfolgt. Nach Abtrennen einer Antennengeißel (die beiden proximalen Segmente bleiben intakt) findet man degenerierte Nervenfortsätze in den Glomeruli des ipsilateralen Deutocerebrum. Bei Calliphora laufen viele dieser Axone über eine Bahn dorsal des Oesophagus in die Glomeruli des contralateralen Deutocerebrum. Ein großer Ast des Antennennerven Calliphora zieht vorbei an der Region der Glomeruli in posteriore Regionen des Oberschlundganglions. Nach Abtrennen der ganzen Antenne einschließlich der proximalen Segmente findet sich in diesem Trakt eine große Zahl degenerierter Axone. Die Synapsen in der Region der Glomeruli ähneln anderen Synapsen, wie sie im Zentralnervensystem von Insekten bisher beschrieben wurden.

Ultrastructure of growth cones in the cerebellar cortex of the neonatal rat and cat

SummaryThe ultrastructure of axonal and dendritic growth cones has been examined in the cerebellar cortex of 7 days old rats and 12 days old cats. The unique feature is a bulge of the perikaryon surface or a varicosity of the growing tip of nerve processes. These cone-like areas contain large amounts of tubular smooth surfaced endoplasmic reticulum (SR) and large vacuoles. They are further characterized by filopodia (Tennyson, 1970) with a fibrillary matrix. Early cell contacts with synaptic membrane specializations are described between filopodia of mossy fiber endings and dendritic growth cones of granular cells. Synaptic vesicles appear early in synaptogenesis. While both vesicles and SR tubules are confined to separate areas of the axonal growth cone it was found that a common affinity to the ZIO staining agent exists. In contrast, the neurofilaments and microtubular components as well as the growth cone vacuoles remain consistently ZIO negative.

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.

Intramembranous particles at the nodes of Ranvier of the cat spinal cord: A morphometric study

Size and distribution of intramembranous particles at nodes of Ranvier of the cat spinal cord were investigated by the freeze-etching technique and compared with those at the internodal axon. The particles are larger (up to 20 nm) at the nodal than at the internodal segment (up to 13 nm), and these large particles are more densely packed in the nodal (400 per sq micrometer) than in the internodal E (external) face (4 per sq micrometer). The nodal E face reveals a much denser overall population (1200--1300 per sq micrometer) of particles than the internodal E face (100--200 per sq micrometer), while at the P (protoplasmic) face the particle density is similar in nodal and internodal segments (1200--1600 per sq micrometer). It is suggested that the large nodal particles may be related to the mechanism of nerve excitation.

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.