CONFIGURATION OF A FILAMENTOUS NETWORK IN THE AXOPLASM OF THE SQUID (LOLIGO PEALII L.) GIANT NERVE FIBER

High-resolution electron microscopy is integrated with physicochemical methods in order to investigate the following preparations of the giant nerve fibers of the squid (Loligo pealii L.): (1) Thin sections of fibers fixed in four different fixatives; (2) fresh axoplasm stained negatively in solutions of different pH and composition; (3) chemically isolated threadlike elements of the axoplasm. A continuous, three-dimensional network can be identified in all these preparations of the axoplasm. The network is composed of coiled or looped unit-filaments ~30 A wide. The unit-filaments are intercoiled in strands ~ 70–250 A wide. The strands are oriented longitudinally in the axoplasm, often having a sinuous course and cross-associations. Microtubules are surrounded by intercoiled unit-filaments and filamentous strands. Calcium ions cause loosening and disintegration of the network configuration. UO₂⁺⁺ ions of a 1% uranyl acetate solution at pH 4.4 display a specific affinity for filamentous protein structures of the squid giant nerve fiber axoplasm, segregating the filamentous elements of the axoplasm in a coiled, threadlike preparation. The uranyl ions combine probably with the carboxyl groups of the main amino acids of the protein—glutamic and aspartic acids. It is proposed that by coiling/decoiling and folding/unfolding of the unit-filaments, shifts in physicochemical properties of the axoplasm are maintained.     

Ultrastructure of the nodes of Ranvier and their surrounding structures in the central nervous system

Summary The nodes of Ranvier and their surrounding structures have been studied by means of serial and single ultrathin sections of the frog's optic tract and diencephalon.The following aspects of the ultrastructure of the myelin sheath at the nodes are described: (a) the site and manner of termination of the compact myelin and glia-satellite cytoplasm; (b) the reflection of the glia-satellite cytoplasm at the node; (c) the formation of compact myelin during development; (d) the involvement of the glia-satellite cell in the metabolism and impulse conduction of the fiber.The nodes are surrounded by a considerable extension of the extracellular space — the perinodal extracellular space or perinodal matrix. The ground substance of the perinodal matrix consists of ill defined granules arranged in patches or in fibrillar or vacuolar manner. Microvilli-like protuberances of the glia and nervous processes emerge into the perinodal matrix. The shape and the volume of the perinodal extracellular space is determined using a reconstruction method from a series of sections through the node in diencephalon.In the diencephalon nerve endings show close contact with the node and its surroundings. These nerve endings contain synaptic vesicles, mitochondria and small opaque particles. Small opaque particles, up to the present not recognized as components of synapses, have been observed in a number of nerve endings in the diencephalon. The possibility is considered that such nervous configurations at the node could be involved in subliminal interactions between different neurons.Based on the ultrastructural data the concept of nodal apparatus is introduced as a working hypothesis. The nodal apparatus consists of the node, terminal compartments of the glia-satellite cell, the perinodal matrix, and the surrounding glia and nervous structures, which may be involved in the nodal activities. The structural pattern of such a nodal apparatus may vary in different parts of the central nervous system indicating the possibility of variation in the functioning of the corresponding nodes.This investigation was supported by a grant from the Medical Research Council, Canada (MA-1247).The author is very much indebted to Mrs. H. Rushforth for excellent technical assistance, to Mr. H. R. A. Meiborg, Groningen, for very skilful printing of the photographs and to Mr. Hoekstra, Groningen, who made the drawing of Fig. 8.     

Die Innervation der Drüsenzellen der Pars distalis der Hypophyse bei der Ente

Zusammenfassung Es wird bei der Ente an Hand von Bielschowsky-Gros-Präparaten die Innervation der Drüsenzellen der Pars distalis der Hypophyse beschrieben. Beinahe in allen Teilen der Pars distalis wird ein dichter nervöser Endplexus in den Drüsenzellsträngen gefunden. Jede einzelne Drüsenzelle steht in unmittelbarem Kontakt mit feineren und gröberen Nervenfasern des Plexus. Diese nervöse Formation wird als eine morphologische Basis für die aus bisherigen experimentellen Arbeiten gefolgerte Wirkung des Nervensystems auf die Bildung und Abgabe der Hormone in der Pars distalis interpretiert und ihr die Übertragung von Nervenimpulsen auf die einzelnen Drüsenzellen zugeschrieben.Daneben wird auch die Endformation des peripheren vegetativen Nervensystems — der sympathische Grundplexus (Boeke) —, die sich zwischen den Drüsenzellsträngen und Sinuscapillaren in der Pars distalis ausbreitet, beschrieben.Beide nervösen Strukturen werden mit den argyrophilen Bindegewebsmembranen in der Pars distalis verglichen und der prinzipielle Unterschied des morphologischen Gefüges beider Gewebsarten festgestellt.Die vorliegenden Ergebnisse wurden auf der LVIII. Tagung der Nederlandse Anatomen Vereeniging am 11. Dezember 1954 in Amsterdam vorgetragen (Nederl. Tijdschr. Geneesk. 1955)     

Hereditary Nephropathy with Hematuria (Alport's Syndrome)

Among 82 members and four generations of a French-Canadian family, 14 cases of hereditary nephropathy (Alport''s syndrome) were documented. Five additional members of the family had died, probably because of this same illness. Deafness occurred in five family members with nephropathy and in one without renal disease. Ten of 12 affected males died in uremia before they had reached the age of 40 years. One of seven affected females died following a pregnancy. In two surviving patients, special investigations failed to elicit intrinsic tubular defects such as amino-aciduria, renal tubular acidosis, hyperphosphaturia or renal glucosuria. Systemic illness such as abnormal aminoacids in serum, primary hyperoxaluria, diabetes mellitus and infections were also excluded. Immunological defects were not demonstrable and the staining of renal biopsy tissue with fluorescein-labelled anti-β₁c, anti-IgG and antifibrinogen was negative. Renal tissue material of early, advanced and terminal hereditary nephropathy showed both tubular and interstitial, vascular and glomerular lesions. Electronmicroscopy showed marked thickening of tubular and glomerular basement membranes, increase of mesangial tissue and fusion of foot processes but failed to demonstrate “immune deposits.” It is postulated therefore that hereditary nephropathy results from an inborn error of metabolism where an as yet unidentified metabolite damages the renal tissue as well as the acoustic nerve, analogous perhaps to the action of certain drugs, e.g. nephro-ototoxic antibiotics.     

Neuronal transformations in Alzheimer's disease

Summary Brains of nine early and four advanced Alzheimer patients have been investigated, utilizing three approaches to specify the threshold state of Alzheimer's disease (AD). Extensive thin sectioning electron microscopy (EM) of frontal lobe biopsies, correlated with stringent clinical assessment, has demonstrated that the neuronal cytoskeleton undergoes specific transformations into paired helical filament-like (PHF-like) strands, which lead to the formation of the insoluble paracrystalline paired helical filaments (PHFs). The neurofilamentous network (NFN) transformation plays an important role in this process, whereby segregation, posttranslational modifications and reassembly of the modified components through autocrosslinking, and phase transition occur. According to our data, the threshold state can be defined as the state of irreversible segregation and posttranslational modification of the NFN and the microtubule-associated proteins. At this state, therapeutic intervention to reverse the disease process may be possible. The results indicate similarities between the formation of the paracrystals of the PHFs and the formation of the tropomyosin-like crystals of the Hirano bodies. Close relationships among PHFs and smooth endoplasmic reticulum and plasma membrane do exist. Enveloped virus-like particles have been observed in neurons containing PHFs. A possible role of these virus-like particles as an etiological agent for AD is discussed.     

Purification and characterization of squid brain myosin.

Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.     

Axial and radial filamentous components of the neurofilamentous network

Summary The three-dimensional structure of the neurofilamentous network of the giant axon of the squid has been investigated by stereo electron microscopy and optical image analysis. The neurofilamentous network with its structural associations intact was partially purified by means of extraction of extruded axoplasm in a physiological buffer. The authors employed a double-grid mounting technique for critical point drying of axoplasm which provides high contrast preparations having great depth suitable for the analysis of spatial relations. There is a striking improvement in the perception of the continuity and three-dimensionality of the network, particularly in thin areas produced by teasing apart the specimen prior to mounting. Sidearms and cross-bridges are readily identifiable.In 1-m thick preparations of the extracted axoplasm the neurofilamentous network is composed of two structural entities: (i) long 10-nm wide filaments approximately parallel with the long axis of the extracted axoplasmic cylinder (axial), (ii) and short, finer filaments projecting from them as sidearms or crossbridges (radial). Optical analysis of micrographs of extracted axoplasm indicates that the radial filamentous components of the neurofilamentous network are distributed predominantly in the range of 32° to 67° with respect to the long axis of the axial filaments. We tentatively assign the 220,000 mol wt peptide observed by SDS-PAGE in this preparation to the radial filaments and the 68,000 mol wt peptide to the axial filaments.     

SPATIAL PATTERNS OF THREADLIKE ELEMENTS IN THE AXOPLASM OF THE GIANT NERVE FIBER OF THE SQUID (LOLIGO PEALII L.) AS DISCLOSED BY DIFFERENTIAL INTERFERENCE MICROSCOPY AND BY ELECTRON MICROSCOPY

The giant nerve fiber of the squid (Loligo pealii L.) has been investigated in situ, and in fresh and fixed preparations, by differential interference microscopy and electron microscopy. A continuous, three-dimensional network, composed of threadlike elements, was disclosed in the axoplasm. The threadlike elements in the axoplasm are twisted as a whole into a steep, right-handed helix. In a peripheral ectoplasmic region, the elements are more parallel to one another and more densely packed than in a central endoplasmic core. The threadlike elements can be resolved into a hierarchy of decreasing order of size. Successive levels of the hierarchy are formed by the association of smaller elements into larger ones. The following levels in the hierarchy of network elements have been distinguished: 1–3-µ-wide threads, 0.1–0.35-µ-wide strands, and 70–250-A-wide unit-filament strands. The differential interference microscope selects, from the network, threads oriented at a specific angle to the long axis of the axon. The specific angle depends upon the orientation of the long axis of the axon relative to the direction of shear. It is postulated that the network configuration is expressed in the solid-state properties of the axoplasm essential for the normal functioning of the nerve fiber.