Fine structure of ependymal cells in the median eminence of the frog and mouse revealed by freeze-etching

Summary Freeze-etched preparations of the ventricular surfaces of ependymal cells clearly reveal the presence of pinocytotic vesicles opening into the third ventricle and large vacuoles formed by broad cell projections. The density of the vesicular openings is approximately 20 per m². The ependymal cells in the median eminence of the frog are adjoined by tight junctions comprised of five to eight interconnected junctional strands, whereas near the median eminence in the mouse only one to two such strands form the tight junction of the ependymal cells. Gap junctions between the adjacent ependymal cells are detected near the median eminence in the mouse but not in the frog.This study was supported in part by a grant from the Japanese Ministry of Education (No. 067670)     

The ultrastructure of the human fetal pineal gland

Summary The innervation of the pineal gland, the cell junctions in this organ and junctions between ependymal cells in the pineal recess were investigated in 27 human fetuses (crown-rump length 30–190 mm).Free nerve boutons containing clear and a few dense core vesicles were present in the pineal parenchyma and in the perivascular spaces. The boutons did not make synaptic contacts with the pinealocytes. No evidence for the presence of noradrenaline in the vesicles of nerve boutons was found.Gap junctions, intermediate-like junctions and desmosomes were frequently seen between the pinealocytes. Ruthenium red was used in three fetuses as an extracellular marker.The continuous endothelial cells surrounding the capillary lumen were connected by tight junctions. This indicates the presence of a blood-brain barrier.Tight junctions were present between the ependymal cells in the pineal recess. These junctions constitute an extracellular barrier between the pineal and the cerebrospinal fluid. Acknowledgements: The author wishes to thank Inger Ægidius and Jb Machen for their technical, Ruth Fatum for her linguistic and Karsten Bundgaard for his photographical assistance     

Interneuronal and glial-neuronal gap junctions in the lamina ganglionaris of the compound eye of the housefly,Musca domestics

Summary The cell-body layer of the lamina ganglionaris of the housefly, Musca domestica, contains the perikarya of five types of monopolar interneuron (L1–L5) along with their enveloping neuroglia (Strausfeld 1971). We confirm previous reports (Trujillo-Cenóz 1965; Boschek 1971) that monopolar cell bodies in the lamina form three structural classes: Class I, Class II, and midget monopolar cells. Class-I cells (L1 and L2) have large (8–15 m) often crescentshaped cell bodies, much perinuclear cytoplasm and deep glial invaginations. Class-II cells (L3 and L4) have smaller perikarya (4–8 m) with little perinuclear cytoplasm and no glial invaginations. The midget monopolar cell (L5) resides at the base of the cell-body layer and has a cubshaped cell body. Though embedded within a reticulum of satellite glia, the L1–L4 monopolar perikarya and their immediately proximal neurites frequently appose each other directly. Typical arthropod (-type) gap junctions are routinely observed at these interfaces. These junctions can span up to 0.8 m with an intercellular space of 2–4 nm. The surrounding nonspecialized interspace is 12–20 nm. Freezefracture replicas of monopolar appositions confirm the presence of -type gap junctions, i.e., circular plaques (0.15–0.7 m diam.) of large (10–15 nm) E-face particles. Gap junctions are present between Class I somata and their proximal neurites, between Class I and Class II somata and proximal neurites, and between Class II somata. Intercartridge coupling may exist between such monopolar somata. The cell body and proximal neurite of L5 were not examined. We also find that Class I and Class II somata are extensively linked to their satellite glia via gap junctions. The gap width and nonjunctional interspace between neuron and glia are the same as those found between neurons. The particular arrangement and morphology of lamina monopolar neurons suggest that coupling or low resistance pathways between functionally distinct neurons and between neuron and glia are probably related to the metabolic requirements of the nuclear layer and may play a role in wide field signal averaging and light adaptation.     

The mature mesonephric nephron of the rabbit embryo

Summary In freeze-fracture replicas of the entire cross-fractured mesonephros of 18 day rabbit embryos the basolateral and luminal cell faces of the different nephron segments were studied and compared with their metanephric counterparts. In the proximal tubule, the shallow zonula occludens exhibited only 1–2 strands and resembled the corresponding metanephric zonula, a very leaky type, which was found with a considerable paracellular flow component in sites of isotonic reabsorption. Gap junctions were restricted to the proximal tubule and were seen more frequently in its terminal segment. The distal tubule harboured two types of tight junctions. The most common type, a band of 5–8 closely parallel strands, matched the zonula occludens of the metanephric straight distal tubule. The observed particle density of the basolateral membrane (2,500±328/m²) was less than that of the proximal tubule (2,642±306). In addition, the collecting tubule exhibited a zonula occludens of the tight variety similar to that which occurred in the metanephric collecting duct. Rod-shaped particles of the luminal membrane were mainly concentrated in some of the intercalated cells but also had developed on principal cells, and occasionally, in the distal tubule. The Wolffian duct, with a deep tight zonula occludens, had an obviously rather inactive epithelium with no conspicuous transport-linked membrane specializations.     

Ultrastructure of arterioles in the cat brain

Summary A total of 110 arterioles were examined in the brains of cats; different sites were studied including the cortex, putamen, pons and crus cerebri. No internal elastic laminae were seen in the subendothelial space, although occasional fragments of elastic material were present in the larger arterioles. The media was composed of one, two or three layers of smooth muscle cells which interlocked in such a way that the vessel wall thickness was constant. Numerous tight junctions were seen between adjacent smooth muscle cells and between the endothelium and smooth muscle cells. Apart from the usual cell organelles, the smooth muscle cells of arterioles had numerous dense patches on the cell surface. The structure of the adventitia varied according to the diameter of the vessel and the site in the brain; it contained adventitial cells, bundles of collagen fibres and nerve fibres. Innervation of arterioles was more constant in the brain stem than in the cortex. Metarterioles had less specialised, atypical smooth muscle cells, a discontinuous media and numerous, extensive myoendothelial tight junctions; they were not innervated by nerve fibres. The diameter of metarterioles was less than 10 m whereas that of arterioles was 10–45 m. The possible functional aspects of arteriolar innervation are discussed.     

Gap junctions are non-randomly distributed inDrosophila wing discs

Summary The distribution of gap junctions in mature larvalDrosophila melanogaster wing discs was analyzed by means of quantitative electron microscopy. Gap junctions are non-randomly distributed in the proximal-distal disc axis and in the apical-basal cell axis of the epithelium. In the epithelial cells, the surface density, number and length of gap junctions are greatest in the apical cell region and distal disc region. The average gap junction surface density is 0.0572 m^–1 and 2.77% of the lateral cell surface is composed of gap junctions. In the adepithelial cells, the gap junction surface density is 0.0005 m^–1 and 0.06% of the cell surface is composed of gap junctions. No gap junctions were observed between epithelial cells and adepithelial cells. The absolute area of gap junctions was estimated in a proximal-distal strip of cells in the disc and is considerably less in the folded regions of the epithelium compared to the flat notum and wing pouch regions. The results are discussed with respect to pattern formation and growth control in imaginal discs.     

Estrogen-dependent changes in the functional interrelationships among neurons,ependymal cells and glial cells of the arcuate nucleus

Summary The synchronizing effect of ethinylestradiol (4 g/g b.w.) on neurons of the arcuate nucleus 700–950 m caudal to the posterior edge of the optic chiasma was studied by karyometry in 6-week-old albino mice during proestrus.The caudal portion of the arcuate nucleus was identified as the most estrogen-sensitive subdivision; all neurons showed an increase in their nuclear area (mean transect, profile area of the nucleus) 1 h following administration of ethinylestradiol. This hypothalamic region was selected for the subsequent electron-microscopic cytometric study to analyze functional interrelationships among neurons, ependymal cells and glial cells. Six and 12 days after ovariectomy no significant change in the nuclear area of neurons and ependymal cells was found 850–950 m behind the posterior slope of the optic chiasma, but the neurons exhibited a decrease in the number of polyribosomes, the volume fraction (V_Vmi) and the surface density of the inner membrane of mitochondria (S_Vmi). A similar decrease in V_Vmi and S_Vmi was measured in the apical part of ependymal cells and in the pericapillary profiles of ependymal and glial cells, which was accompanied by a reduction in the surface density of ependymal processes extending into the ventricular lumen. In addition, no change of V_Vmi and S_Vmi was seen in the basal subnuclear part of ependymal cells.This bipolar functional reaction of ependymal cells after ovariectomy is discussed as an indicator of ependymal control of neuronal activity by sequestering biologically active agents, e.g., transmitters of neurohormones, in their apical and basal extensions facing the ventricular surface or the pericapillary space.     

Effect of α-naphthaleneacetic acid and sucrose levels on the development of cultured embryos of coconut

Mature coconut embryos were germinated in a modified Murashige and Skoog medium and then cultured on BMY₃ medium incorporating sucrose in the range of 4 to 8%. -Naphthaleneacetic acid (NAA) was added into the medium at concentrations ranging from 0 to 800 M for periods of 4 to 24 weeks. Application of NAA for 4 weeks stimulated shoot growth, whereas application periods greater than 4 weeks had no significant effect. NAA in the range of 100–300 M stimulated elongation of the primary root and the optimum concentration increased with increases in sucrose levels. Production of adventitious roots was stimulated by the addition of NAA with levels of 200 M and above being the most effective. Increasing the sucrose concentration from 4% through to 8% stimulated root elongation in the absence of NAA and inhibited shoot growth whether NAA was present or absent.Abbreviations IAA indole-acetic acid - NAA -naphthaleneacetic acid     

Neuronal connections in the ocellus of the wasp (Paravespula vulgaris L.)

Summary Studies of the dorsal ocelli of the wasp Paravespula vulgaris (L.) led to the following results: Under a biconvex corneal lens, 150 m in thickness, about 600 receptor cells are located. The rhabdomeres of two adjacent cells form a closed plate-like rhabdom (0.5–1.0 m in thickness, 6 m in width and 10–25 m in depth or length).In the lateral ocellus the receptor cells synapse up to 8 ocellar nerve fibers, and in the median ocellus they synapse up to 16 (20–30 m thick) ocellar nerve fibers.The ocellar synaptic plexus may display three types of synapses between the two types of neurons: (i) Receptor-cell axons are presynaptic to dendrites of the first-order interneurons. (ii) Dendrites of the first-order interneurons are presynaptic to receptor-cell axons. (iii) The subunits of a dendrite of first-order interneurons form synapses with each other.The present work was partially supported by the Stiftung Volkswagenwerk     

Micromatabilin, a new biliverdin conjugate in the spider,Micromata rosea (Sparassidae)

Summary Micromatabilin, the green pigment ofMicromata rosea, is characterized as a mixture of biliverdin conjugates. The two isolated fractions have identical absorption bands (free base: 620–630 m, hydrochloride: 690 m, zinc complex: 685–690 m). Chromic acid degradation yields imides I, II, IIIa, and IIIb. Differences in the non-hydrolytic degradation and in polarity lead to the conclusion that fraction 1 is a monoconjugate and fraction 2a diconjugate of biliverdin.     

Distribution and fine structure of ependymal cells possessing intracellular cysts in the aqueductal wall of the rat brain

Summary The wall of the cerebral aqueduct was examined in 20 male rats at the light- and electron-microscopic levels. Disorder in ciliary orientation was occasionally seen in ordinary ependymal cells. Ependymal cells possessing intracellular cysts of 5 to 30 urn in diameter were observed within and beneath the aqueductal ependyma in all animals examined. Light-microscopic reconstruction from serial, 10-m thick frontal sections revealed an extensive distribution of cystic ependymal cells (CECs), especially along the ependymal ridges in the rostral half of the aqueduct, and along the dorsal region of the aqueductal lining in the caudal half. Both cystic and surface membranes of CECs bore microvilli and cilia. Ectopic ependymal cells (EECs) characterized by densely packed microvilli, well-developed intermediate junctions and cilia, but without cysts, were situated in the subependymal region adjacent to a CEC or another EEC. The ependymal ridges were long, narrow and sporadically stratified ependymal linings extending rostrocaudally and bilaterally along the aqueductal surface. Tanycyte-like cells filled the surface region of the ridge, and CECs and EECs were frequently seen in the core. Intraventricularly injected microperoxidase was detected among densely packed microvilli but not in the cystic lumina of CECs, indicating that EECs and CECs are distinct entities.     

Fenestration of the epithelium lining the roof of the fourth cerebral ventricle in amphibia

Summary Scanning electron microscopy of the caudal end of the roof of the fourth cerebral ventricle in four amphibian species shows that numerous pores occur between the ependymal cells. These pores have diameters ranging from 5–100 m; they permit bulk flow of cerebrospinal fluid out of the ventricular system into the subarachnoid space.     

Ex Vitro Phenotype Stability is Affected by In Vitro Cultivation

Plant phenotype stability during ex vitro growth, one of the main requirements of plant micropropagation, was tested on tobacco. Plants cultivated in vitro in the presence of 3 % sucrose under photon flux density (PFD) of 200 mol m^–2 s^–1 (3 % HL plants) showed the best growth and photosynthetic parameters in the course of 7-day acclimation. However, significant change in phenotype of these plants appeared under a decrease in PFD to 50 mol m^–2 s^–1 during further ex vitro growth (in the period of 7^th – 17^th day). Much higher internodia elongation was found in 3 % HL plants in comparison with plants grown in vitro on sucrose media under PFD of 50 mol m^–2 s^–1 (3 % LL) or without sucrose either under PFD of 50 mol m^–2 s^–1 or 200 mol m^–2 s^–1 (0 % LL, 0 % HL). It can be presumed that 3 % HL plants show permanent demand for high PFD. Neither ABA or chlorophyll contents nor de novo thylakoid membrane synthesis were related to the morphogenic effect of low PFD. Changeable contents of hexoses in leaves of 3 % HL and 3 % LL plants were in no direct correlation to the elongated growth.     

Gap junctions of the muscles of the small and large intestine

Summary The distribution of gap junctions (nexuses) in various parts of the small and large intestines of the guinea-pig was studied using the freeze-fracture technique and in thin sections. The percentage area of smooth muscle cell surface occupied by gap junctions varies from 0.50% in the circular muscle of the duodenum to zero in the longitudinal muscle of the ileum. In the circular muscle of the jejunum and ileum the area occupied by nexuses is 0.22% (or about 11 m² per cell). The sizes of junctions range from less than 0.01 m² to 0.20 m², with two-thirds of them being smaller than 0.05 m². In the colon, gap junctions are rare, very small and confined to the circular muscle layer. Even the smallest aggregates of intramembrane particles correspond to areas of close apposition between the membranes of adjacent cells; it is therefore justified to interpret them as being gap junctions. Some gap junctions are formed between a smooth muscle cell and an interstitial cell. Gap junctions are not found in the longitudinal muscle of the small intestine; this is in sharp contrast to the abundance of gap junctions in the adjacent circular layer.In the small intestine of cats and rabbits, gap junctions are abundant in the circular muscle layer, whereas they are very small in size and very few in number in the longitudinal muscle layer.The authors wish to thank Mr Peter Trigg and Miss Eva Franke for help and support. This work was supported by grants from the Medical Research Council and the Central Research Fund of the University of London     

Injection of foreign particles and their intracellular translocation in living phloem fibers

Summary Droplets of castor oil 5–10 in diameter were injected into individual cells of living differentiated phloem fibers. The droplets retained their integrity and their movement was recorded on 16-mm movie film. These foreign particles were translocated, along with indigenous particles, by the rotational cytoplasmic currents of the fibers.     

Ultrastructural details of Sertoli cell junctional complexes in vivo and their modifications in tissue culture

Summary This study was carried out using thin sections, lanthanum tracing, and freeze-cleaving in order to investigate the Sertoli cell junctions of rat testis in vivo and their maintenance in culture. The presence of a new type of membrane specialization has been revealed. This consists of a close association between tight junctions and nexuses. The Sertoli cell contact specializations show a progressive disorganization in vitro correlated with the duration of the period in culture. The relationship between the morphological changes in Sertoli cell junctions and the lack of spermatogenesis in culture is discussed.Research performed under the C.N.R. project Biology of Reproduction     

Electrical coupling among heart cells in the absence of ultrastructurally defined gap junctions

Summary Cells from the ventricles of 7-day chick embryos were aggregated into spheroidal clusters by 48 hr of culture on a gyratory platform. All aggregates beat spontaneously and rhythmically. Microelectrode impalement of widely separated cells within aggregates indicated that they were coupled, as evidenced by a mean coupling ratio (V ₂/V ₁) of 0.81±0.09, and by simultaneity of intrinsic electrical activity (action potentials and subthreshold voltage fluctuation). In freeze-fracture preparations, the cell surfaces contained numerous small groups of intramembrane protein (IMP) particles, arranged in macular clusters, and linear and circular arrays. Using the criterion of 4 clustered IMP particles to define a minimal gap junction, 0.27% of the total P-face examined was devoted to gap junctional area. Within such clusters particles were packed at about 8200/m²; in nonjunctional regions, particles were scattered at a density of about 2000/m². When exposed to cycloheximide (CHX: 50g/ml) for 24–48 hr, coupling ratio declined to 0.44. This decrease could be attributed largely to leakiness of the nonjunctional membrane. Aggregates continued to beat rhythmically and in a coordinated fashion even after 72 hr in inhibitor. However, between 3–21 hr in CHX gap junctional area declined to 0.10%, and all particle clusters disappeared from the P-faces of aggregates in CHX for 24 or 48 hr. Neither macular nor linear particle arrays were seen. We conclude that organized gap junctions are unnecessary for electrotonic coupling between embryonic heart cells. These findings support the idea that low-resistance cell-to-cell pathways may exist as isolated channels scattered throughout the area of closely apposed plasma membranes.     

Surface charge of eosinophils. Binding of cationic particles and measurement of cellular electrophoretic mobility

Summary The surface charge of eosinophils, isolated from the peritoneal exudate of rats by the use of a Metrizamide gradient, was analysed by ultrastructural cytochemistry and cellular electrophoretic mobility. Binding of colloidal iron hydroxide and of cationized ferritin particles at pH 1.8 and 7.2 respectively, was observed on the surface of the eosinophils. An electrophoretic mobility of –1.08 and –1.39 m·s^–1·V^–1·cm was determined for living and glutaraldehyde-fixed eosinophils, respectively. Treatment of the cells with neuraminidase reduced the electrophoretic mobility to –0.64 m·s^–1·V^–1·cm (glutaraldehyde-fixed), reduced significantly and abolished completely the binding of both colloidal iron hydroxide and cationized ferritin particles to the surface of the cells. These results indicate that sialic acid exists on the surface of eosinophils, where it accounts for part of the negative surface charge.     

Distribution and activity of bacteria in the headwaters of the Rhode River Estuary,Maryland, USA

A transect along the axis of the headwaters of a tidal estuary was sampled for microbial, nutrient, and physical parameters. Chlorophylla averaged 42g 1^–1 and phytoplankton comprised an estimated 80% of the total microbial biomass as determined by adenosine triphosphate (ATP). Bacterial concentrations ranged from 0.3–53.9×10⁶ cells ml^–1 and comprised about 4% of the total living microbial biomass. Bacterial production, determined by³H-methyl-thymidine incorporation was about 0.05–2.09× 10⁹ cells 1^–1 h^–1, with specific growth rates of 0.26–1.69 d^–1. Most bacterial production was retained on 0.2m pore size filters, but passed through 1.0m filters. Significant positive correlations were found between all biomass measures and most nutrient measures with the exception of dissolved inorganic nitrogen nutrients where correlations were negative. Seasonal variability was evident in all parameters and variability among the stations was evident in most. The results suggest that bacterial production requires a significant carbon input, likely derived from autotrophic production, and that microbial trophic interactions are important.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy