Ciliated neurons and different types of synapses in anterior hypothalamic nuclei of reptiles

Summary The magnocellular paraventricular and supraoptic nuclei and the parvocellular preoptic and periventricular nuclei have been studied by light and electron microscopy in Emys orbicularis, Lacerta agilis and Elaphe longissima. The ultrastructure of cerebrospinal fluid (CSF)-contacting neurons was described in the preoptic and periventricular nuclei of Emys and Lacerta species. Single 9×2+0 cilia similar to those of the CSF-contacting dendritic terminals were found on perikarya of non CSF-contacting nerve cells, in all four investigated nuclei. The cilia project from funnel-like invaginations of the perikarya into the intercellular space. In the neurons of the nuclei studied, granular vesicles were found, their size being mainly 1,600 Å in the paraventricular nucleus, about 1,800 Å in the supraoptic nucleus, 1,100 Å in the periventricular nucleus and 800 Å, or up to 1,250 Å in the preoptic nucleus. In general, the neurons possess synapses of the axo-somatic, axo-somatic spine, axo-dendritic and axo-dendritic spine types. In the supraoptic nucleus, multiple interdigitated synapses were observed. Presynaptically, either synaptic vesicles only, or synaptic vesicles and dense core vesicles of different sizes (600 to 800 Å, about 1,100 Å, 1250 Å, and up to 2,000 Å) were found. It is discussed whether the above described 9×2+0 cilia may represent some kind of hypothalamic sensory structure that earlier physiological studies postulated to exist. The ciliated hypothalamic perikarya are considered by the authors to be a more differentiated form of the CSF-contacting neurons. The different types of synapses indicate multilateral connections of the nerve cells of the nuclei studied.Dedicated to Prof. Dr. Berta Scharrer on the occasion of her 70th birthday     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

Histology and ultrastructure of the neurohypophysis of the south american lungfish,Lepidosiren paradoxa

Summary The neurohypophysis of the South American lungfish Lepidosiren paradoxa has been studied with light and electron microscopy, including the Falck-Hillarp technique for catecholamines. The pars nervosa hypophyseos is a well-marked, dorsally located subdivision of the pituitary gland composed of lobes or follicles, each one constituted of a central core of ependymal cells, a subependymal hilar region made up of nerve fibers and a peripheric palisade zone of nerve endings which contact capillary vessels. Four types of neurosecretory axons can be distinguished under the electron microscope. Type I, the most common, contains spherical elementary granules of high electron density, 1500–1800 Å in diameter. The scarce type II axons contain irregularly-shaped elementary granules. Type III contains only small clear vesicles, 400–600 Å in diameter. Type IV, mostly present in regions of the gland contacting the pars intermedia, contain large granulated vesicles, 900–1000 Å in diameter. The Falck-Hillarp technique revealed axons with a positive reaction for catecholamines at sites corresponding approximately to the location of type IV of the electron microscope.Ependymal cells are of large size, linking the cerebrospinal fluid, the nerve endings and the blood vessels. A conspicuous membrane-bound, spherical dense material, 1400–2000 Å in diameter, is observed in both the apical and vascular processes of these cells. The ependymal processes which traverse the hilar and palisade regions contain structures resembling degenerated neurosecretory axons. These results are discussed in relation with the currently available information on the comparative anatomy of the pars nervosa. The possible functional significance of ependymal cells and of each type of axon are also discussed.This study was aided by the following grants: NIH NS 06953 to Prof. De Robertis, Consejo Nacional de Investigaciones Científicas y Técnicas to Prof. Zambrano, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires and Comisión de Investigaciones Cientificas de la Universidad Nacional de la Plata: to Prof. Iturriza.The authors are indebted to Prof. De Robertis for his generosity in granting us his laboratory facilities, and to Dr. F. J. J. Risso and Mr. A. Fernández (Resistencia, Chaco) who provided the specimens used in this study. The able microtechnical assistance of Miss L. Riboldazzi and Mrs. R. Raña and the photographic work of Mr. A. Saenz are much appreciated.Members of the Scientific Career, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

What can we learn about the lipid vesicle structure from the small-angle neutron scattering experiment?

Small-angle neutron scattering (SANS) on the unilamellar vesicle (ULV) populations (diameter 500 and 1,000 Å) in D₂O was used to characterize lipid vesicles from dimyristoylphosphatidylcholine (DMPC) at three phases: gel L_β′, ripple P_β′ and liquid L_α. Parameters of vesicle populations and internal structure of the DMPC bilayer were characterized on the basis of the separated form factor (SFF) model. Vesicle shape changes from nearly spherical in the L_α phase to elliptical in the P_β′ and L_β′ phases. This is true for vesicles prepared via extrusion through pores with the diameter 500 Å. Parameters of the internal bilayer structure (thickness of the membrane and the hydrophobic core, hydration and the surface area of the lipid molecule) were determined on the basis of the hydrophobic–hydrophilic (HH) approximation of neutron scattering length density across the bilayer ρ(x) and of the step function (SF) approximation of ρ(x). DMPC membrane thickness in the L_α phase (T=30°C) demonstrates a dependence on the membrane curvature for extruded vesicles. Prepared via extrusion through 500 Å diameter pores, vesicle population in the L_α phase has the following characteristics: average value of minor semi-axis 266±2 Å, ellipse eccentricity 1.11±0.02, polydispersity 26%, thickness of the membrane 48.9±0.2 Å and of the hydrophobic core 19.9±0.4 Å, surface area 60.7±0.5 Ų and number of water molecules 12.8±0.3 per DMPC molecule. Vesicles prepared via extrusion through pores with the diameter 1,000 Å have polydispersity of 48% and membrane thickness of 45.5±0.6 Å in the L_α phase. SF approximation was used to describe the DMPC membrane structure in L_β′ (T=10°C) and P_β′ (T=20°C) phases. Extruded DMPC vesicles in D₂O have membrane thickness of 49.6±0.5 Å in the L_β′ phase and 48.3±0.6 Å in the P_β′ phase. The dependence of the DMPC membrane thickness on temperature was restored from the SANS experiment.     

Ultrastructure of the neurosecretory system of the Colorado potato beetle,Leptinotarsa decemlineata (Say)

Summary The ultrastructure of seven types of neurosecretory cells (NSC) in the medial and lateral groups of the protocerebrum is described. The differences among cell types established earlier by light microscopy parallel differences in size and appearance of the neurosecretory particles observed in electron micrographs. No relationship was found between the affinity for Gomori's paraldehyde fuchsin stain and the nature of the particles.The secretions of the A-, A₁-, and C-types of NSC of the medial group are characterized by electron-dense neurosecretory granules of 1250 Å dia., medium-dense granules of 2100 Å, and electron-lucent vesicles of 1700 Å, respectively. The L-type NSCof the lateral group contain smaller (1300 Å) or larger (1700 Å) neurosecretory granules. The medial B- and E-types of NSC and the lateral L_B-type contain granulated vesicles (1200 Å) of the same appearance. These cell types differ in other respects and most likely have separate functions.The author wishes to thank the Laboratory of Virology of the Agricultural University for the use of the electron microscope, Mr. J. Groenewegen and Miss J. van Rinsum for technical assistance, and Professor J. Lattin for correcting the English text. Part of the work has been done while the author was in the service of the Netherlands Organization for the Advancement of Pure Research (ZWO, grant 942-48), and the National Council for Agricultural Research (TNO).     

The ultrastructure of organelles appearing in spermatids and nutritive cells of Cipangopaludina malleata

Summary The ultrastructure of organelles appearing in the early typical and atypical spermatids, and the nutritive cells of Cipangopaludina malleata has been examined by a Siemens' electron microscope Elmiskop I.Mitochondria appearing in the early typical spermatid have doughnut-like profiles in which the internal ridges appear as triple-layered membranes arranged radially and extending into the interior of the organelle without reaching the other side. Each membrane 40–60 Å in width, separated by a clear interspace 60–90 Å wide, is characterized by a porous structure 20–30 Å in diameter which suggests a filtration apparatus for enzymes.Walls of the flattened saccules consisting the Golgi apparatus are calculated 35–60 Å thick, in which an electron-lucent, porous structure about 30 Å wide has been revealed.The smooth-surfaced endoplasmic reticulum is bordered by a triple-layered membrane consisting of two opaque layers with a less opaque interspace 20–30 Å wide. The outer membrane ca. 15 Å wide presents a more linear appearance than the dotted arrangement of the inner membrane 20–25 Å thick.The plasma membrane is composed of a triple-layered structure where two dense lines 15 Å wide are separated by a layer 20–30 Å thick of less density.The electron micrographs for the present studies were taken with the Siemens electron microscope, model Elmiskop I, at the Anatomical Institute of Kiel University, Germany. The one of the authors, G. Yasuzumi is deeply grateful to Prof. Dr. W. Bargmann and Dr. A. Knoop for the privilege of using this instrument and other equipments in the Laboratory.     

Contrast variation studies of clathrin coated vesicles by small-angle neutron scattering

Structural information on clathrin coated vesicles has been obtained by small angle neutron scattering using contrast variation. A characteristic peak in the neutron scattering profile, which is apparent in 75 % D₂O, as well as in H₂O, disappears when contrast matching the protein component of the coated vesicles in 42% D₂O. Neutron, as well as dynamic, light scattering give a coated vesicle size of about 900 Å in H₂O and D₂O, but for neutron scattering the diameter decreases when matching out the protein coat of the clathrin coated vesicles. From the match point for the clathrin coated vesicles it is demonstrated that the clathrin cages do contain internal membrane. The mass of 34 MD and composition of 75% protein and 25% lipid found from the analysis of the small-angle scattering data are both in good agreement with the values reported in the literature. Electron microscopy gives an average outer diameter of 880 Å for the coated vesicles and an average diameter of 460 Å for the vesicle itself. Offprint requests to: Correspondence to: R. Bauer     

Substructure of membranes in cultivated chick embryo fibroblasts

Summary Electron microscope examination of the plasma membrane of chick embryo fibroblasts cultured in vitro revealed the presence of a single osmiophilic layer about 90 Å thick and a substructure composed of ovoid sub-units associated with an amorphous component. These ovoid sub-units measured approximately 112 Å along the major axis and 75 Å along the minor axis and were composed of a central core, approximately 30 Å by 60 Å, surrounded by a peripheral component.Examination of other membranous components of these cells revealed a similar ovoid subunit structure in a single layered membrane. Differences in thickness and in the sizes of ovoid sub-units were seen in these membranes. The ergastoplasmic membranes, the outer nuclear membranes, the outer mitochondrial and the Golgi membranes were found to be the thinnest.These varied in thickness from approximately 75 Å to 80 Å. The thickest membranes seen were the inner nuclear membranes. These were approximately 100 Å thick. The dimensions of the ovoid sub-units corresponded with differences in the thickness of the various membranes. These findings support the concept of a particulate substructure of cell membranes.This work was aided by Research Grant PH 5593 from the National Science Foundation. Some of the equipment used was purchased with funds from the National Institutes of Health Grant 2TI GM 326. I wish to thank Dr. Robert M. Dougherty from the Department of Microbiology who grew and supplied me with the chick embryo fibroblast cultures used in these studies, and Mrs. Ursula Feller fer her technical assistance.     

The fine structure of the retinal plexus in Octopus vulgaris

Summary The plexiform layer of the octopus retina was investigated by means of electron microscopy. The axonic processes of visual cells contain closely packed microtubules, 300 Å in diameter, running parallel to one another along the long axis of the processes. Visual cells also send out a large number of thin axon collaterals. Each of them forms presynaptic knobs with numerous clear vesicles along their course. These are assumed to be concerned with the reciprocal communication between visual cells. Nerve endings with dense-cored vesicles form synaptic contacts with visual cells. The visual cells show some spherical protrusions into the perivascular spaces.Octopuses, captured off the coast of Onagawa (Miyagi-ken, Japan) in autumn of 1963 and 1964, were offered for this research through the kindness of Dr. Kyoji Tasaki, Assistant Professor of Physiology in Tohoku University School of Medicine.I wish to thank Professor Toshiyuki Yamamoto for his encouragement and suggestions throughout all stages of this work, and also Mr. Masae Kato for his technical assistance in drawing.     

Synaptic relationships in the plexiform layers of carp retina

Summary The synaptic contacts made by carp retinal neurons were studied with electron microscopic techniques. Three kinds of contacts are described: (1) a conventional synapse in which an accumulation of agranular vesicles is found on the presynaptic side along with membrane densification of both pre- and postsynaptic elements; (2) a ribbon synapse in which a presynaptic ribbon surrounded by a halo of agranular vesicles faces two postsynaptic elements; and (3) close apposition of plasma membranes without any vesicle accumulation or membrane densification.In the external plexiform layer, conventional synapses between horizontal cells are described. Horizontal cells possess dense-core vesicles about 1,000 Å in diameter. Membranes of adjacent horizontal cells of the same type (external, intermediate or internal) are found closely apposed over broad regions.In the inner plexiform layer ribbon synapses occur only in bipolar cell terminals. The postsynaptic elements opposite the ribbon may be two amacrine processes or one amacrine process and one ganglion cell dendrite. Amacrine processes make conventional synaptic contacts onto bipolar terminals, other amacrine processes, amacrine cell bodies, ganglion cell dendrites and bodies. Amacrine cells possess dense-core vesicles. Ganglion cells are never presynaptic elements. Serial synapses between amacrine processes and reciprocal synapses between amacrine processes and bipolar terminals are described. The inner plexiform layer contains a large number of myelinated fibers which terminate near the layer of amacrine cells.This work was supported by an N.I.H. grant NB 05404-05 and a Fight for Sight grant G-396 to P.W. and N.I.H. grant NB 05336 to J.E.D. The authors wish to thank Mrs. P. Sheppard and Miss B. Hecker for able technical assistance. P.W. is grateful to Dr. G. K. Smelser, Department of Ophthalmology, Columbia University, for the use of his electron microscope facilities.     

Cerebrospinal fluid-contacting neurons,ciliated perikarya and “peptidergic” synapses in the magnocellular preoptic Nucleus of teleostean fishes

Summary The magnocellular preoptic nucleus of fishes (Anguilla anguilla, Amiurus nebulosus, Cyprinus carpio, Carassius auratus, Ctenopharyngodon idella, Cichlasoma nigrofasciatum) has been studied by light and electron microscopy.Two kinds of neurons were found: a) large, electron-dense, Gomori-positive cells with moderate acetylcholinesterase (AChE) positivity which contain granulated vesicles of 1400 to 2200 Å (in average 1600 to 1800 Å), and b) small, strongly AChE-positive, electron-lucent neurons containing granulated vesicles of 900 to 1200 Å. The nerve cells are supplied with axo-somatic and axo-dendritic synapses. These are formed by axon terminals containing either 1. synaptic vesicles of 500 Å, or 2. synaptic vesicles of 500 Å and dense-core vesicles of 600 to 800 Å, or 3. synaptic vesicles of 600 Å and granulated vesicles of up to 1100 Å, or 4. synaptic vesicles of about 400 Å and granulated vesicles of up to 1800 Å. The presence of peptidergic and numerous other synapses shows the complexity of the organization and afferentation of the magnocellular preoptic nucleus.In the eel, both types of nerve cells form dendritic terminals within the cerebrospinal fluid (CSF). These CSF contacting dendrites are supplied with 9×2+0 cilia. In the other species investigated, only some large neurons build up intraventricular endings. The ependymofugal process of the CSF contacting neurons enters the preoptic-neurohypophysial tract.Perikarya of both the large and the small cells may give rise to single, paired or multiple 9×2+0 cilia extending into the intercellular space. The number of CSF contacting neurons is reciprocal to the number of perikarya with intercellular cilium. These latter cells may represent modified, more differentiated forms of the CSF contacting neurons. We think that atypical cilia protruding into the intercellular space may have the same significance for the intercellular fluid as the cilia of the intraventricular dendrites of the CSF contacting neurons for the CSF.Dedicated to Prof. Dr. W. Bargmann on the occasion of his 70th birthday.     

Pigment containing lipid vesicles

Summary Vesicles obtained by sonication of chlorophylla-lecithin mixtures dispersed in an aqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophylla. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophylla molecule per 55 lecithins can be incorporated into the membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102±5 Å and independent of the chlorophylla content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Electron microscopic study on the innervation of the pancreas of the domestic fowl

Summary The innervation of the pancreas of the domestic fowl was studied electron microscopically. The extrapancreatic nerve is composed mostly of unmyelinated nerve fibers with a smaller component of myelinated nerve fibers. The latter are not found in the parenchyma. The pancreas contains ganglion cells in the interlobular connective tissue. The unmyelinated nerve fibers branch off along blood vessels. Their synaptic terminals contact with the exocrine and endocrine tissues. The synaptic terminals can be divided into four types based on a combination of three kinds of synaptic vesicles. Type I synaptic terminals contain only small clear vesicles about 600 Å in diameter. Type II terminals are characterized by small clear and large dense core vesicles 1,000 Å in diameter. Type III terminals contain small clear vesicles and small dense core vesicles 500 Å in diameter. Type IV terminals are characterized by small and large dense core vesicles. The exocrine tissue receives a richer nervous supply than the endocrine tissue. Type II and IV terminals are distributed in the acinus, and they contact A and D cells of the islets. B cells and pancreatic ducts are supplied mainly by Type II terminals, the blood vessels by Type IV terminals.This work was supported by a scientific research grant (No. 144017) and (No. 136031) from the Ministry of Education of Japan to Prof. M. Yasuda     

Calcium-induced fusion of didodecylphosphate vesicles: The lamellar to hexagonal II (HII) phase transition

Summary Electron microscopic techniques have been employed to investigate the ability of didodecylphosphate vesicles (diameter approx. 900 Å) to fuse in the presence of Ca²⁺. As revealed by negative staining, Ca²⁺ induces extensive fusion and large vesicles with diameters up to 7000 Å are formed. In a processsecondary to fusion, the fused vesicles display a tendency to flatten and are subsequently transformed into extended tubular structures. Freeze-fracture electron microscopy, in conjunction with³¹P NMR and selected area electron diffraction measurements indicate that the tubes are packed in a hexagonal (H_II) array and that the amphiphiles are converted from the lamellar to the hexagonal H_II phase.The relationship between membrane fusion and the lamellar-to-hexagonal phase transition is discussed in terms of formation and abundance of transiently stable inverted micellar intermediates at contact regions between two interacting membranes. A model for the conversion of the (vesicular) lamellar into the (tubular) hexagonal H_II phase is presented, taking into account the molecular shape of the amphiphile. The relevance of using simple synthetic amphiphiles as models for phospholipid bilayers and complex biomembrane behavior is briefly discussed.     

The fine structure of neuromuscular junctions and contact zones between body wall muscle cells of Ascaris lumbricoides (var. suum)

Summary Neuromuscular junctions and close membrane apposition between body wall muscle cells of Ascaris lumbricoides (var. suum) have been examined with the light and electron microscopes. It was found that the body wall muscle cells send out elongate processes from their basal, myofibril containing portion to terminate on dorsal and ventral nerves. When observed with the aid of the electron microscope the neuromuscular junctions were seen to consist of several muscle cell processes in apposition to a single axon. The intersynaptic cleft was approximately 350–500 Å wide. Both the axolemma and sarcolemma were triple layered membranes which were 75–80 Å thick. Electron dense patches were observed at intervals on the apposed membranes which were due to increased thickness of the inner membrane leaflets of axolemma and sarcolemma. Muscle cell membranes, at the level of the neuromuscular junction, were in close apposition resulting in an apparently five-layered membrane complex which was 170–210 Å thick. The sarcolemmata in these regions were separated by 10–50 Å. Presynaptic axons contained mitochondria, microtubules which were 180–270 Å in diameter, and two, morphologically distinct types and sizes of synaptic vesicles. One was 200–600 Å in diameter, with a single, triple-layered membrane bounding a center of low electron density. The other was 600–1200 Å in diameter, with a single, triple-layered membrane bounding a central, electron dense granule of 500–800 Å size.The functional significances of the close membrane appositions between body wall muscle cells and of the two types of synaptic vesicles found at the neuromuscular junctions of Ascaris lumbricoides were discussed with respect to their possible role in neuromuscular physiology.Supported by U.S.P.H.S. Grant No. NB-01528 and Research Career Development Award No. 9-K3-NB-15255. — The author wishes to express his grateful appreciation for the excellent technical assistance given by Miss Gabrielle Rouiller during the course of this investigation.     

Degeneration of monoamine nerves in anterior byssus retractor muscle of Mytilus induced by 5,6-dihydroxytryptamine

Summary Preliminary ultrastructural studies on the effects of 5,6-Dihydroxytryptamine (5,6-DHT) on the anterior byssus retractor muscle (ABRM) of Mytilus show degeneration of 2 types of monoaminergic nerves after 10 days of drug treatment. One type contained large granular vesicles (560–1,680 Å) while the other had small granular vesicles (200–640 Å). These axons may possibly represent serotonergic and dopaminergic nerves, thought to innervate this muscle.Two other types of profiles seemed to be unaffected by the drug. One conforms to cholinergic nerves while the other has a predominance of large opaque vesicles (1,200–2,500 Å). The significance of these findings is discussed in the light of recent observations on the neurotoxic effects of 5,6-DHT on vertebrate and molluscan nerves.The author is grateful to Professor G. Burnstock for research facilities and Professor B. M. Twarog for advice and encouragement. This work was supported by the Ramaciotti Foundation     

Structure of vesicles formed from non-ionic dialkylglycerol poly(oxyethylene) ether surfactants: effect of electrolyte and cholesterol

Five non-ionic dialkylglycerol poly(oxyethylene) ether surfactants, designated 2C_mE_n (where m, the number of carbons in each alkyl chain = 16 or 18, and n, the number of oxyethylene units = 12, 16 or 17) have been examined for their ability to form vesicles when dispersed in water or in an aqueous solution of 154 mM NaCl, alone or in the presence of 50 mol% cholesterol. Freeze fracture electron microscopy and light scattering showed that regardless of the hydrating fluid, all the non-ionic surfactants, with the exception of 2C₁₆E₁₇ and 2C₁₈E₁₇, formed vesicles in the absence of cholesterol – 2C₁₆E₁₇ and 2C₁₈E₁₇ instead formed micellar aggregates. All surfactants, however, formed vesicles in the presence of 50 mol% cholesterol. Small angle neutron scattering studies of the surfactant vesicles enabled the bilayer thickness and repeat distance (d-spacing) to be determined. The bilayers formed by all the non-ionic surfactants in the absence of cholesterol were surprisingly thin (∼50 Å for the E₁₂ containing surfactants and ∼64 Å for 2C₁₈E₁₆) most likely due to the intrusion of oxyethylene groups into the hydrophobic core of the bilayers. In contrast, however, the non-ionic surfactants exhibited a relatively large d-spacing of around ∼130–150 Å. The addition of 50 mol% cholesterol had a dramatic effect on the thickness of the vesicle bilayer, increasing its size by 10–20 Å, most probably because of an extrusion of oxyethylene from the hydrophobic region of the bilayer and/or a reduction in the tilt on the surfactant alkyl chains. Additionally the presence of cholesterol in a vesicle tended to reduce slightly both the d-spacing and the thickness of the water layer separating the bilayers. The presence of NaCl, even at the low concentrations used in the study, did affect the properties of the bilayer such that it reduced the d-spacing and, in the case of cholesterol-containing systems, also reduced bilayer thickness.     

Electron microscopic observations on synapse-like contacts between pituicytes and different types of nerve fibers in the anuran pars nervosa

Summary Pituicytes of Rana pipiens could be classified into two types, pale and dense, according to their relative densities of cytoplasm and the populations of free ribosomes and cell organelles. An intermediate type of pituicyte was also recognized.Lipid droplet such as are typical in the cytoplasm of mammalian pituicytes, are not in the cytoplasm of either types of frog pituicyte. Both types have long cytoplasmic processes which run among the nerve fibers, and some of them end at the pericapillary space.Nerve endings making synapse-like contacts with the cell bodies or the processes of the pituicyte are frequent. According to the structures and sizes of granules and vesicles in the nerve endings, these endings are classified into one of three types: 1) A, which appears to be a peptidergic neuronal ending containing dense granules 1,200–2,000 Å in diameter and small clear vesicles 300–400 Å in diameter; 2) B, which appear to be monoaminergic endings containing cored vesicles 600–1,000 Å in diameter and small clear vesicles 300–500 Å in diameter; 3) C, which appear to be cholinergic endings containing only small clear vesicles. Type C endings are relatively rare. In the synaptic area the axonal membranes appose those of the pituicytes across a gap of about 200 Å and numerous presynaptic vesicles are clustered or accumulated near the presynaptic membranes.The author wish to express his hearty thanks Professor Dr. A. Gorbman, Zoology Department, University of Washington, Seattle, U.S.A. and Professor Dr. H. Fujita for their helpful advices and criticisms. The frog tissues were obtained and fixed in Professor A. Gorbman's laboratory supported by U.S.P.H.S. grant NS 04887.     

On the fine structure of the frog's rod outer segments,observed by the freeze-etching technique

Summary The fine structure of the frog's (Rana esculenta) rod outer segments was investigated by two different methods: most of the experiments were made by means of the freeze-etching technique. The replicas were then examined by electron microscopy (40,000 X).By means of a second method, rod outer segments were negatively stained prior to electron microscopy.Inspection of the electron micrographs revealed that the frog's rod outer segments seem to be built up of three groups of elongated structures interpreted as fibrils (Fäden) arranged regularly at approximately equal distances. The diameters of the fibrils are below 100 Å; they depend on the state of light adaptation and on the chemical preparation before freeze-etching. The fibrils partly cross each other. In addition, there were found four groups of approximately equal distances between the fibrils. The order of magnitude of these spacings is from about 50 Å to a few hundred Å.Negatively stained outer segments also reveal fibrils. The results are expressed in a working hypothesis consisting of two parts. It is supposed first that the core of the rod outer segment represents a three dimensional paracrystalline lattice (Raumgitter) of three different types of fibrils (d ₁, d₂, d₄). The distances between the fibrils are interpreted as the lattice constants (a ₁, a₂, a₃, a₄). A unit cell of the lattice would consist of a web (Geflecht) of two different types of fibrils (d ₁, d₂) and four layers of parallel fibrils of the third type (d ₄).It is supposed, secondly, on the basis of a volume-evaluation, that the d₁-fibrils contain rhodopsin, those of type d ₂ another protein (not rhodopsin), and fibrils of type d ₄ lipids.The working hypothesis is supported by experimental findings of other authors (obtained by negative staining and diffraction of light and X-rays).Attempts have been made to relate some electron micrographs of ultrathin sections to those of replicas. (Rosenkranz et al., 1969; Rosenkranz, 1969a.)I wish to thank Prof. Dr. H. Stieve for the interest he took in this work through critical discussions and financial support. I also wish to thank Prof. A. Ruthmann, Ph. D., for introducing me to electron microscopy and for his linguistic aid. That Prof. Dr. K. Mühlethaler, ETH Zürich, and Prof. Dr. F. Schwanitz, KFA Jülich, put their freeze-etching apparatus and electron microscope at my disposal is gratefully acknowledged. The technical assistance of Miss M. Deichmann is also acknowledged.     

Determination of bilayer thickness and lipid surface area in unilamellar dimyristoylphosphatidylcholine vesicles from small-angle neutron scattering curves: a comparison of evaluation methods

Small-angle neutron scattering (SANS) experiments were performed on unilamellar 1,2-dimyristoylphosphatidylcholine (DMPC) vesicles prepared in heavy water by extrusion through polycarbonate filters with 500 Å pores. The data obtained at 30±0.1 °C were evaluated using a five-strip function model of the bilayer coherent neutron scattering length density, three different approximate form factors describing scattering from vesicles, and different methods of evaluation of the experimental data. It is shown that the results obtained from the SANS data in the range of scattering vector values 0.0316 Å^–1<q<0.0775 Å^–1 are not sensitive to the vesicle form factor, nor to the evaluation method. Using the hollow sphere model of vesicles convoluted with the Gaussian distribution of their sizes, a constrained bilayer polar region thickness of 9 Å and a DMPC headgroup volume of 325.5 ų, it was possible to obtain from the experimental data the DMPC surface area as 58.9±0.8 Ų, the bilayer thickness as 44.5±0.3 Å and the number of water molecules as 6.8±0.2 per DMPC located in the bilayer polar region.