Morphology and Electrophysiological Properties of Squid Giant Axons Perfused Intracellularly with Protease Solution

Squid giant axons were perfused intracellularly with solutions containing various kinds of proteases (1 mg/ml). Except for a 10 µ layer inside the axolemma the axoplasm was removed by a 5 min perfusion with Bacillus protease, strain N' (BPN'). The resting and action potentials were unchanged and the axon maintained its excitability for more than 4 hr on subsequent enzyme-free perfusion. After perfusion with protease solution for 30 min the axoplasm was almost completely removed. The excitability was maintained, but the action potential became prolonged and rapidly developed a plateau of several hundred milliseconds. The change was not reversible even when the enzyme was removed from the perfusing fluid. Two other enzymes, prozyme and bromelin, also removed the protoplasm without blocking conduction. Trypsin suppressed within 3 min the excitability of the axon. It is suggested that the proteases alter macromolecules in the excitable membrane and thus affect the shape of the action potential.     

Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus

Giant axons of the spiny lobster, Panulirus argus, are filled with microtubules that are decorated with fine, irregular filaments. Mitochondria and membrane-limited clear vesicles are the only other distinguishable elements in the axoplasm and are located around the periphery of the axon near the axolemma. Neither 100 A neurofilaments nor 70 A microfilaments are evident in fixed, intact axons or in negatively stained axoplasm. Actin-like microfilaments are a prominent constituent of the glial cells that closely ensheathe the axons, and gel electrophoresis studies suggest that most of the actin in the nerve fibers is located in the glia rather than in the axons. Studies of isolated axoplasm indicate that microtubules are the primary elements stabilizing the axoplasm. The microtubules in the isolated axoplasm are disrupted by Ca2+ in the medium in the presence of protease inhibitors.     

Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma

In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.     

Three-dimensional reconstruction of the 14-filament fibers of hemoglobin S.

The supramolocular structure of hemoglobin S has been studied by electron microscopy and computer-based image reconstruction. Negatively stained fibers prepared by the lysis of sickled cells or the stirring of hemoglobin S hemolysates have been observed to be almost exclusively of the 20-nm diameter form. These fibers have a periodic variation in diameter between the extremes of 18 nm and 23 nm. Computed Fourier transforms of the fibers show a, highly complex pattern of reciprocal space maxima, with 30 maxima on 20 layer-lines clearly resolved. The Bessel orders of the maxima were assigned with the aid of a newly developed technique, a combined real-space Fourier-space reconstruction method (REFORM). This method utilizes the filtered image produced by the inverse Fourier transform of the low-resolution maxima to calculate in real space the crosssection of a helical fiber. The REFORM analysis indicated that the fibers have an elliptical cross-section and are composed of 14 hexagonally packed filaments with 10 outer filaments surrounding four inner filaments. On the basis of this cross-section, the Bessel orders of all the maxima were assigned, permitting the calculation of three-dimensional reconstructions by Fourier Bessel synthesis. From these reconstructions details of the location of hemoglobin S molecules of each filament were obtained. Hemoglobin S molecules are staggered in adjacent filaments to produce a closely packed helical structure. Reconstructions of fibers at various stages of disassembly revealed a stable intermediate containing 10 filaments which could be characterized in terms of the loss of two pairs of specific outer filaments. A partially disassembled fiber with only six filaments at positions corresponding to three inner and three outer filaments of the parent structure was also identified. The six-filament structure appears to be produced from the 10-filament structure by the loss of two specific pairs of filaments. Thus pairs of filaments are evidently significant structural units in the stabilization of the complete fibers and the orientation of the molecules in these pairs may be related to the filament pairs known to occur in crystals of hemoglobin S.     

Subaxolemmal cytoskeleton in squid giant axon. I. Biochemical analysis of microtubules, microfilaments, and their associated high-molecular- weight proteins

Using the squid giant axon, we analyzed biochemically the molecular organization of the axonal cytoskeleton underlying the axolemma (subaxolemmal cytoskeleton). The preparation enriched in the subaxolemmal cytoskeleton was obtained by squeezing out the central part of the axoplasm using a roller. The electrophoretic banding pattern of the subaxolemmal cytoskeleton was characterized by large amounts of two high-molecular-weight (HMW) proteins (260 and 255 kD). The alpha, beta-tubulin, actin, and some other proteins were also its major constituents. The 260-kD protein is known to play an important role in maintaining the excitability of the axolemma (Matsumoto, G., M. Ichikawa, A. Tasaki, H. Murofushi, and H. Sakai, 1983, J. Membr. Biol., 77:77-91) and was recently designated "axolinin" (Sakai, H., G. Matsumoto, and H. Murofushi, 1985, Adv. Biophys., 19:43-89). We purified axolinin and the 255-kD protein in their native forms and further characterized their biochemical properties. The purified axolinin was soluble in 0.6 M NaCl solution but insoluble in 0.1 M NaCl solution. It co-sedimented with microtubules but not with actin filaments. In low-angle rotary-shadowing electron microscopy, the axolinin molecule in 0.6 M NaCl solution looked like a straight rod approximately 105 nm in length with a globular head at one end. On the other hand, the purified 255-kD protein was soluble in both 0.1 and 0.6 M NaCl solution and co-sedimented with actin filaments but not with microtubules. The 255-kD protein molecule appeared as a characteristic horseshoe-shaped structure approximately 35 nm in diameter. Furthermore, the 255-kD protein showed no cross-reactivity to the anti-axolinin antibody. Taken together, these characteristics lead us to conclude that the subaxolemmal cytoskeleton in the squid giant axon is highly specialized, and is mainly composed of microtubules and a microtubule-associated HMW protein (axolinin), and actin filaments and an actin filament-associated HMW protein (255-kD protein).     

The pattern of cell wall deterioration in lignocellulose fibers throughout enzymatic cellulose hydrolysis

Cell wall deterioration throughout enzymatic hydrolysis of cellulosic biomass is greatly affected by the chemical composition and the ultrastructure of the fiber cell wall. The resulting pattern of cell wall deterioration will reveal information on cellulose activity throughout enzymatic hydrolysis. This study investigates the progression and morphological changes in lignocellulose fibers throughout enzymatic hydrolysis, using (transmission electron microscopy) TEM and field emission scanning electron microscopy (FE‐SEM). Softwood thermo‐mechanical pulp (STMP) and softwood bleached kraft pulp (SBKP), lignocellulose substrates containing almost all the original fiber composition, and with lignin and some hemicellulose removed, respectively, was compared for morphology changes throughout hydrolysis. The difference of conversion between STMP and SBKP after 48 h of enzymatic hydrolysis is 11 and 88%, respectively. TEM images revealed an even fiber cell wall cross section density, with uneven middle lamella coverage in STMP fibers. SKBP fibers exhibited some spaces between cell wall and lamella layers due to the removal of lignin and some hemicellulose. After 1 h hydrolysis in SBKP fibers, there were more changes in the fiber cross‐sectional area than after 10 h hydrolysis in STMP fibers. Cell wall degradation was uneven, and originated in accessible cellulose throughout the fiber cell wall. FE‐SEM images illustrated more morphology changes in SBKP fibers than STMP fibers. Enzymatic action of STMP fiber resulted in a smoother fiber surface, along with fiber peeling and the formation of ribbon‐disjunction layers. SBKP fibers exhibited structural changes such as fiber erosion, fiber cutting, and fiber splitting throughout enzymatic hydrolysis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Characterization of phospholipase A2 and acyltransferase activities in squid (Loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions.

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.

The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.

Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

A re-evaluation of the cytology of cat Pacinian corpuscles

Summary The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.     

The possible role of fixed membrane surface charges in acetylcholine release at the frog neuromuscular junction

Summary Bass and Moore [Proc. Nat. Acad. Sci. 55:1214 (1966)] proposed that the vesicles containing acetylcholine undergo Brownian motion in the nerve terminals. Acetylcholine is released whenever a vesicle touches the inner face of the axolemma of the nerve terminal. The frequency at which contact is made is limited by an energy barrier that must be overcome before the vesicle can touch the axolemma. The energy barrier has two components. (1) An electrostatic repulsion between positive, fixed charges on the vesicles and a relatively positive potential at the face of the axolemma that is generated by the resting potential. (2) A layer of water molecules held to the vesicle by the surface charge. This model is inconsistent with experimental data. A modification of the model is presented. Both the vesicle and the inner face of the axolemma are assumed to have fixed, negative surface charges that are responsible for the energy barrier. By a series of simplifications, the model leads to two predictions. (1) A plot of the ln (miniature end plate potentials/sec) as a function of the concentration of ions in the axoplasm)^–0.5 should give a straight line. (2) A plot of ln (end plate potential amplitudes) as a function of (extracellular Ca⁺⁺ concentration)^–0.5 should give a straight line. These predictions are shown to agree reasonably well with experimental data.     

Carbon nanotube reinforced Bombyx mori silk nanofibers by the electrospinning process

Nanocomposite fibers of Bombyx mori silk and single wall carbon nanotubes (SWNT) were produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun into nanofibers. The morphology, structure, and mechanical properties of the electrospun nanofibers were examined by field emission environmental scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and microtensile testing. TEM of the reinforced fibers shows that the single wall carbon nanotubes are embedded in the fibers. The mechanical properties of the SWNT reinforced fiber show an increase in Young's modulus up to 460% in comparison with the un-reinforced aligned fiber, but at the expense of the strength and strain to failure.     

Molecular structure of the axolemma of developing axons following altered gliogenesis in rat optic nerve

Axonal and axolemmal development of fibers from rat optic nerves in which gliogenesis was severely delayed by systemic injection of 5-azacytidine (5-AZ) was examined by freeze-fracture electron microscopy. In neonatal (0-2 days) rat optic nerves, all fibers lack myelin, whereas in the adult, virtually all axons are myelinated. The axolemma of neonatal premyelinated fibers is relatively undifferentiated. The P-fracture face (P-face) displays a moderate (approximately 550/micron 2) density of intramembranous particles (IMPs), whereas the E-fracture face (E-face) has few IMPs (approximately 125/micron 2) present. By 14 days of age, approximately 25% of the axons within control optic nerves are ensheathed or myelinated, with the remaining axons premyelinated. The ensheathed and myelinated fibers display increased axonal diameter compared to premyelinated axons, and these larger caliber fibers exhibit marked axonal membrane differentiation. Notably, the P-face IMP density of ensheathed and myelinated fibers is substantially increased compared to premyelinated axolemma, and, at nodes of Ranvier, the density of E-face particles is moderately high (approximately 1300/micron 2), in comparison to internodal or premyelinated E-face axolemma. In optic nerves from 14-day-old 5-AZ-treated rats, few oligodendrocytes are present, and the percentage of myelinated fibers is markedly reduced. Despite delayed gliogenesis, some unensheathed axons within 5-AZ-treated optic nerves display an increased axonal diameter compared to premyelinated fibers. Most of these large caliber fibers also exhibit a substantial increase in P-face IMP density. Small (less than 0.4 micron) diameter unensheathed axons within treated optic nerves maintain a P-face IMP density similar to that of control premyelinated fibers. Regions of increased E-face particle density were not observed. The results demonstrate that some aspects of axolemma differentiation continue despite delayed gliogenesis and the absence of glial ensheathment, and suggest that axolemmal ultrastructure is, at least in part, independent of glial cell association.     

Electron microscope observations on the submicroscopic organization of the retinal rods

The submicroscopic organization of the retinal rods of the rabbit has been studied with high resolution electron microscopy in thin longitudinal and cross-sections. The outer rod segment consists of a stack of flattened sacs or cisternae each of them limited by a thin homogeneous membrane of about 30 A. The membrane of the rod sacs is attached to the surface membrane and is also in continuity with short tubular stalks of about 100 to 150 A which apparently end in relation with the connecting cilium. The bundle of filaments that constitute the connection between the outer and the inner segments is described under the name of connecting cilium. This fibrous component has a structure that is very similar to that of the cilium. It shows 9 pairs of peripheral filaments of about 160 A in diameter, a matrix material, and a surface membrane. Very infrequently two central single filaments are observed. The connecting cilium has a typical basal body in the inner segment; its distal end penetrates the outer segment, where it establishes some structural relation to the rod sacs. The relationships and submicroscopic organization of the connecting cilium were studied in longitudinal and in cross-sections passing at different levels of the rod segments. The inner rod segment shows two distinct regions: a distal and a proximal one. The distal region, corresponding to the ellipsoid of classical histology is mainly composed of longitudinally packed mitochondria. It also contains the basal body of the cilium, vacuoles of the endoplasmic reticulum, dense particles, and intervening matrix with very fine filaments. In the proximal region of the inner segment the mitochondria are lacking and within the matrix it is possible to recognize elements of the Golgi complex, vacuoles of the endoplasmic reticulum, dense particles and numerous neuroprotofibrils of 160 to 200 A in diameter which collect and form a definite bundle at the exit of the rod fiber. The interpretation of the connecting fibers as a portion of a cilium and of the outer segment as a differentiation of the distal part of a primitive cilium are discussed. The importance of the continuity of the surface membranes of the outer segment, connecting cilium, and inner segment is emphasized and its possible physiological role is discussed.     

Cell cycle-specific changes in the ultrastructural organization of prematurely condensed chromosomes

Prematurely condensed chromosomes (PCC) of HeLa cells synchronized in different phases of the cell cycle were analyzed by high-resolution scanning electron microscopy. The purpose of this study was to examine changes in the arrangement of the basic 30-nm chromatin fiber within interphase chromosomes associated with progression through the cell cycle. These studies revealed that highly condensed metaphase chromosomes and early G1-PCC consisted of tightly packed looping fibers. Early to mid G1-PCC were more extended and exhibited gyres suggestive of a despiralized chromonema. Further attenuation of PCC during progression through G1 was associated with a gradual transition from packed looping fibers to single extended longitudinal fibers. This process occurs prior to the initiation of DNA synthesis which appears to be localized within single longitudinal fibers. Following replication of a chromosome segment, extended longitudinal fibers were rapidly reorganized into packed looping fiber clusters concomitant with the formation of a multifibered chromosome axis. This results in the characteristic “pulverized” appearance of S-PCC when viewed by light microscopy. Subsequently, adjacent looping fiber domains coalesce, resulting in the uniformly packed, looping fiber arrangement observed in G2-PCC. Spiralization of the chromonema during the G2-mitotic transition results in the formation of highly compact metaphase chromosomes.     

Membrane-Associated Cytoskeletal Proteins in Squid Giant Axons

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.     

Active movement in vitro of bundle of microfilaments isolated from Nitella cell

Subcortical fibrils composed of bundles of F-actin filaments and endoplasmic filaments are responsible for endoplasmic streaming. It is reported here that these fibrils and filaments move actively in an artificial medium containing Mg-ATP and sucrose at neutral pH, when the medium was added to the cytoplasm squeezed out of the cell. The movement was observed by phase-contrast microscopy or dark-field microscopy and recorded on 16-mm film. Chains of chloroplasts linked by subcortical fibrils showed translational movement in the medium. Even after all chloroplasts and the endoplasm were washed away by perfusion with fresh medium, free fibrils and/or filaments (henceforth, referred to as fibers) not attached to chloroplasts continued travelling in the direction of the fiber orientation. Sometimes the fibers formed rings and rotated. Chloroplast chains and free fibers or rings continued moving for 5-30 min at about half the rate of the endoplasmic streaming in vivo. Calcium ion concentrations < 10(-7) M permitted movement to take place. Electron microscopy revealed that both fibers and rings were bundles of F-actin filaments that showed the same polarity after decoration with heavy meromyosin.     

Enhancement of the Amazonian Açaí Waste Fibers through Variations of Alkali Pretreatment Parameters

The açaí fruit depulping produces large amounts of long lignocellulosic fiber bundles that are disposed in the environment. Chemical pretreatments may improve açaí fibers favoring their usage in advanced materials. This work aimed to define optimal alkali reaction parameters to improve the properties of açaí fibers. Two NaOH concentrations (5 % and 10 %) and two reaction temperatures (80 °C and 100 °C) were tested. The raw and treated fibers were analyzed by scanning electron microscopy, Fourier transformed infrared spectroscopy, X‐ray diffraction, and thermal analyses. All the alkali pretreatments separated fibers from the bundles, unblocked pit channels by removing silicon structures, exposed the inner lignin, partially removed non‐cellulosic compounds, and raised the cellulose crystalline index. The highest temperature and NaOH content resulted in better cleaning and isolation of the fibers, while milder conditions better preserved the cellulose crystalline structure and thermal stability.     

Human zona pellucida during in vitro fertilization: an ultrastructural study using saponin, ruthenium red, and osmium-thiocarbohydrazide.

The human zona pellucida (ZP) and its changes during in vitro fertilization in oocytes at different maturational stages and polypronuclear ova at one- to four-cells stages were studied by transmission electron microscopy (TEM) and correlative scanning electron microscopy (SEM). To define the microstructure of the ZP, its amorphous masking material was removed using a detergent (saponin), and its structural glycoproteins were stabilized with a cationic dye, ruthenium red, followed by osmium-thiocarbohydrazide treatment. These methods allowed in all samples the clear visualization of variously arranged networks of filaments composing the outer and inner surfaces of the ZP. These filaments were straight or curved, 0.1-0.4 microns in length and 10-14 nm thick as seen via TEM or 22-28 nm thick as seen via SEM (the difference in thickness was due to the presence of the metal coating for SEM). The filament arrangement was remarkably different between the inner and outer surfaces of the ZP and among the various stages studied. The filaments of the outer surface of the ZP were basically arranged in "large" and "tight" meshed networks. Mature oocytes and fertilized (polypronuclear) ova had a regular alternating pattern of wide and tight meshed networks of filaments. On the other hand, immature and atretic oocytes displayed almost exclusively a tight meshed network of filaments. The inner surface filaments of the ZP of unfertilized oocytes at any stage were arranged in repetitive structures characterized by numerous short and straight filaments anastomosing with each other and sometimes forming at the intersections small, rounded structures. After fertilization, the inner surface of the ZP displayed numerous areas where filaments fused together. Collectively, these data clearly reveal that oocyte maturation and fertilization in humans are accompanied by changes of ZP filaments arrangement, which may be relevant in the processes of binding, penetration, and selection of spermatozoa.     

The fine structure of the pigment epithelium and photoreceptor cells of the newt,Triturus viridescens dorsalis (Rafinesque)

The morphology of the retinal pigment epithelium and photoreceptor cells has been studied in the common newt Triturus viridescens dorsalis by light, conventional transmission and scanning electron microscopy. The pigment epithelium is formed by a single layer of low rectangular cells, separated by a multilayered membrane (Bruch's membrane) from the vessels of the choriocapillaris. The scleral border of the pigment epithelium is highly infolded and each epithelial cell contains smooth endoplasmic reticulum, myeloid bodies, mitochondria, lysosomes, phagosomes and an oval nucleus. Inner, pigment laden, epithelial processes surround the photoreceptor outer and inner segments. The three retinal photoreceptor types, rods, single cones and double cones, differ in both external and internal appearance. The newt, rod, outer segments appear denser than the cones in both light and electron micrographs, due to a greater number of rod lamellae per unit distance of outer segment and to the presence of electron dense intralamellar bands. The rod outer segments possess deep incisures in the lamellae while the cone lamellae lack incisures. Both rod and cone outer segments are supported by a peripheral array of dendritic processes containing longitudinal filaments which originate in the inner segment. The inner segment mitochondria, forming the rod ellipsoid, arelong and narrow while those in the cone are spherical to oval in shape. The inner segments of all three receptor cell types also contain a glycogen-filled paraboloid and a myoid region, just outside the nucleus, rich in both rough and smooth endoplasmic reticulum. The elongate, cylindrical nuclei differ in density. The rod nuclei are denser than those of the cones, contain clumped chromatin and usually extend further vitreally. Similarly, the cytoplasm of the rod synaptic terminal is denser than its cone counterpart and contains synaptic vesicles almost twice as large as those of the cones. Photoreceptor synapses in rods and cones are established by both superficial and invaginated contacts with bipolar or horizontal cells.     

Alteration of birefringence signals from squid giant axons by intracellular perfusion with protease solution

The optical signal, arising from a transient birefringence change associated with excitation, was recorded from a squid giant axon together with the membrane potential change, and the effect of removal of the axoplasm on the optical signal was examined. In an unperfused axon, repetitive stimulation at a frequency of about 100 Hz produced two kinds of optical response. The initial response had a brief, spike-like time course and was elicited by each stimulating pulse. The delayed response had a slow time course and the sign of decreased light intensity, and summated with repetitive stimulation. Most of the axoplasm was removed from interior of the axon by intracellular perfusion with solutions containing pronase at a concentration of 0.1 mg/ml. The delayed response could selectively be eliminated by perfusion with a pronase-containing solution for 2–8 min. The result was interpreted as showing that the delayed birefringence signal originates from axoplasm when its gel structure was transiently disturbed by an increased Ca²⁺ influx associated with excitation. When perfusion was further continued the duration of the action potential started increasing and often a prominent after-depolarization appeared. At this stage the initial optical response was again followed by a large show signal with the sign of increased light intensity. This reversed delayed response was tentatively assumed to originate from the membrane with some remaining axoplasm, but its cause is still not understood.     

Structure-function relationship of human spinal ligaments

A combination of light, scanning and transmission electron microscopy was used to investigate the morphology and ultrastructure of normal human spinal ligaments sampled from adult surgical specimens. The ligamenta flava consist mostly of dense elastic fibers, whereas the supraspinous and interspinous ligaments are preponderantly collagenous. In all ligaments, the collagen fascicles are characterized by a regular crimp structure. The inner collagen fibers of interspinous ligaments tend to be oriented parallel to the spinous processes while those of the peripheral layers run in postero-cranial direction. The presence of proteoglycan filaments is clearly demonstrated in all of the ligaments examined. They are mainly located at the d band of the collagen fibrils. These findings are discussed in relation to the function of the posterior ligamentous system. It is suggested that the interspinous ligaments are able to transmit tension from the thoracolumbar fascia to the spine. Finally, the spinal ligaments are thought to be involved in the control mechanism of the spine.