Permeability studies of the guinea pig placental labyrinth

Summary Permeability of the fetal endothelium within the guinea pig placental labyrinth is studied by means of horse-radish peroxidase (HRP) and ionic lanthanum as diffusion tracers. The paracellular transport of HRP is restricted by the occluding junctions of the fetal endothelium. In contrast, ionic lanthanum readily permeates most of the intercellular junctions and rapidly infiltrates the basal lamina. Freeze-fracture replicas reveal zonulae occludentes connecting the fetal endothelial cells. The network of the zonulae occludentes is variable, exhibiting highly complex areas as well as single strand interconnections. A correlation between the permeability studies and freeze-fracture findings is discussed.Supported by grants from the Consejo Nactional de Investigaciones Cientificas y Técnicas (CONICET), the Alexander-von-Hemboldt-Stiftung and the Deutsche ForschungsgemeinschaftResearch Professor of the Alexander-von-Humboldt-Stiftung and Career Investigator of CONICET     

Seasonal changes of the blood-testis barrier in viscacha (Lagostomus maximus maximus): A freeze-fracture and lanthanum tracer study

Adult male viscachas (Lagostomus maximus maximus) were gathered from their natural habitat during the period of complete spermatogenesis (June) and during the month of maximum testicular regression (August). The testes were processed by conventional electron microscopic technique using lanthanum nitrate (electron-dense intercellular tracer) to define the intercellular spaces below the inter-Sertoli tight junctions and by freeze-fracture techniques. During complete spermatogenesis the tracer surrounds spermatogonia, preleptotene, and leptotene spermatocytes and stops at the level of the inter-Sertoli tight junctions below all germ cells displaying synaptonemal complexes (zygotene-pachytene spermatocytes) and germ cells in more advanced stages of differentiation. Conversely, during testicular regression the tracer percolates all intercellular spaces between Sertoli cells and the remaining germ cells (spermatogonia and few preleptotene and leptotene spermatocytes.) During complete spermatogenesis, freeze-fracture replicas exhibit numerous inter-Sertoli tight junction strands parallel to each other and to the basal lamina. During spermatogenesis decay, the inter-Sertoli tight junctions are found to be short, tortuous, frequently interrupted, and often associated with extented membranous areas of gap junctions. © 1993 Wiley-Liss, Inc.     

Electron microscope observations on the formation of primitive villi in rat small intestine with special reference to intercellular junctions.

Formation and fusion of intraepithelial cavities have long been considered an essential process in the histogenesis of the intestinal mucosa. By electron microscope observation of thin sections and freeze-fracture replicas of the small intestine of rat fetuses, we first demonstrated the initial steps in the formation of intraepithelial cavities: A focal tight junction (macula occludens) was formed in the abluminal part of the epithelium, after which the membrane of an intracellular cavity was fused with that of the focal tight junction to form an intercellular (intraepithelial) cavity enclosed by a zonula occludens. The present study also revealed that gap junctions appeared and enlarged simultaneously with the formation of primitive villi and differentiation of absorptive cells. These gap junctions gradually came to be confined in the epithelium of intervillous regions where proliferation and differentiation of epithelial cells took place. Absorptive cells in villi rarely had gap junctions. These results suggest that tight and gap junctions play important roles in the histogenesis of the intestinal mucosa, and in the proliferation and differentiation of epithelial cells.     

Dynamics of gap junctions between horizontal cells in the goldfish retina

Summary Experimental alterations of gap junctions between outer horizontal cells have been demonstrated in freeze-fracture replicas of goldfish retina. The alterations consisted predominantly of an increase of connexon densities and a decrease in the variability of the arrangement of connexons. They were observed i. in dark adapted retinae, ii. in animals with crushed optic nerves, iii. in picrotoxin-and bicuculline-treated animals. Since experiment i. is characterized by a depolarization of the horizontal cell, and experiment iii. was shown by others to result in uncoupling of horizontal cells, we conclude that the functional connectivity of horizontal cells might be correlated with the structure of gap junctions. An interesting detail is the differentiated reaction of axonal and perikaryal gap junctions on dark adaptation or blindness: whereas normally the axonal gap junctions are less densely packed, they increase their connexon density in darkness or blindness much more than the perikaryal gap junctions.     

Membrane specializations in the first optic neuropil of the housefly,Musca domestica L. II. Junctions between glial cells

Summary Membrane specializations between the three types of glial cells in the first optic neuropil (lamina ganglionaris) of the housefly were determined from thin sections and freeze-fracture replicas. Three strata of glia cells are present in the lamina, A relatively thin layer of satellite glia covers the distal (perikaryal) rind of the lamina and these cells wrap retinular axons that enter the lamina. The central synaptic fields of the lamina neurons are enclosed by epithelial glia, while the proximal surface of the lamina is capped by marginal glial cells.Satellite glia bond to each other via desmosomes, septate and gap junctions. Freeze-fracture replicas show gap junctions as aggregations of E face particles and P face pits on the intramembranous surfaces. Parallel rows of P face particles are indicative of septate junctions. Angulated, intersecting, P face particle ridges are arranged in circumferential bands around retinular axons at the glia-axon interface. Thin section correlates of these junctions are presented.Epithelial glia are characterized by elaborate series of parallel membranes which appear to be suspended in the cytoplasm but may be the invaginated plasma membranes of a neighbouring glial cell. An intermembranous cleft of 40–50 Å is noted and this area has an appreciable electron density which gives the appearance of a gap junction. When cleaved, these membranes show plaques of particles on the P face.The marginal glial cells are relatively large and are joined by a newly discovered junction which is characterized (from freeze-fracture data) by numerous, undulating, uninterrupted, parallel P face ridges which sometimes become circular and form enclosures. In thin sections, electron-dense material fills the membrane appositional areas and in tangential sections faint diffuse parallel striae are seen. This specialized cell contact may be a variant of a continuous junction although, based on fracture replicas, there are obvious similarities to tight junctions. These membrane specializations are related, in the three dimensions of the optic cartridges, to functions in a possible blood-eye barrier system.     

Perineurial and glial cells in the tickBoophilus microplus (Acarina: Ixodidae): freeze-fracture and tracer studies

Summary In the cattle tickBoophilus microplus, the cells of the perineurium are characterized by accumulations of glycogen which increase dramatically after feeding. Gap junctions couple both these perineurial cells which ensheath the C.N.S. and the underlying glial cells. No tight junctions have been found between perineurial cells and there is in consequence no blood-brain barrier. Using ionic lanthanum as a tracer the extensive gap junctions are shown to have no occluding effect and lanthanum penetrates through the perineurium and glial layers to the level of the axonal surfaces. By colloidal lanthanum impregnation and freeze-fracture studies, the gap junctions appear to be typical of arthropods in that their particles show a characteristic diameter (13 nm in freeze-fracture), are distributed relatively loosely within the junctional plaques and fracture onto the E face of the junctional membranes. Semi-ordered particle arrays are found on E face membranes of adjacent axons and glia which may represent axoglial junctions.On leave from C.S.I.R.O., Division of Entomology, Australia.     

High-resolution proteomic mapping in the vertebrate central nervous system: close proximity of connexin35 to NMDA glutamate receptor clusters and co-localization of connexin36 with immunoreactivity for zonula occludens protein-1 (ZO-1)

Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for "co-localization" vs. "close proximity" of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina. In goldfish brain, confocal microscopy revealed immunofluorescence for connexin35 (Cx35) and NMDA-R1 (NR1) glutamate receptor protein in Mauthner Cell/Club Ending synapses. By FRIL double labeling, NR1 glutamate receptors were found in clusters of intramembrane particles in the postsynaptic membrane extraplasmic leaflets, and these distinctive postsynaptic densities were in close proximity (0.1-0.3 microm) to neuronal gap junctions labeled for Cx35, which is the fish ortholog of connexin36 (Cx36) found at neuronal gap junctions in mammals. Immunogold labeling for Cx36 in adult rat retina revealed abundant gap junctions, including several previously unrecognized morphological types. As in goldfish hindbrain, immunogold double labeling revealed NR1-containing postsynaptic densities localized near Cx36-labeled gap junction in rat inferior olive. Confocal immunofluorescence microscopy revealed widespread co-localization of Cx36 and ZO-1, particularly in the reticular thalamic nucleus and amygdala of mouse brain. By FRIL, ZO-1 immunoreactivity was co-localized with Cx36 at individual gap junction plaques in rat retinal neurons. As cytoplasmic accessory proteins, ZO-1 and possibly related members of the membrane-associated guanylate kinase (MAGUK) family represent scaffolding proteins that may bind to and regulate the activity of many neuronal gap junctions. These data document the power of combining immunofluorescence confocal microscopy with FRIL ultrastructural imaging and immunogold labeling to determine the relative proximities of proteins that are involved in short- vs. intermediate-range molecular interactions in the complex membrane appositions at synapses between neurons.     

豚鼠海马CAI区锥体细胞间的缝隙连接——超薄切片和冷冻复型的电镜观察

本实验用超薄切片和冷冻复型电镜技术,研究了豚鼠海马CAI区锥体细胞间的缝隙连接(GJ)。在冷冻复型样品中见到,海马CAI区锥体细胞劈裂面EF上有清楚的GJ存在,另外在锥体细胞顶树突EF面上,也见到GJ。上述GJ的膜内微粒聚集成区,微粒的大小和数目不一,有的区内排列成行,较整齐;有的区内排列不整齐或较分散。     

Rapid formation of gap-junction-like structures induced by glycerol

Intercellular junctions, in identified in freeze-fracture by narrowing of the intercellular gap and codistribution of P-face membrane particles, proliferate during incubation of excised rat prostate tissue in 30% glycerol solution. These junctions fulfill the criteria used to identify gap junctions in freeze-fracture replicas with respect to the size and uniformity of their component particles, the codistribution of the particle aggregates on P-faces of adjacent membranes, and the narrowing of the extracellular cleft at the junction. These gap-junction-like structures form on the lateral surfaces of epithelial cells, where they are normally scarce, within minutes after exposure to glycerol. Glycerol-induced junction formation is not blocked by DNP, a metabolic uncoupler, or by cycloheximide, a protein synthesis inhibitor. Newly formed junctions occur initially in clusters and the number per cluster decreases as individual junctions become larger with longer periods of incubation, suggesting that the clusters coalesce. The structural changes that precede and accompany the formation of these junctions at early times of incubation are comparable to the changes reported to precede the formation of gap junctions accompanying hormonal treatment, development, and other means of natural induction.     

Structural modifications of lutein cell gap junctions during pregnancy in the rat and the mouse

By use of lanthanum tracer and freeze-fracture procedures it was found that granulosa-lutein cells of the pregnant mouse and rat ovaries are connected by gap junctions and septate-like zones of contact. Lutein cell gap junctions enlarge and become partially internalized by the end of the first week of gestation. Expansion of the gap junction domain appears to be due initially to intercalation of particles along borders of small gap junctions devoid of smaller non-junctional particles. The number of gap junction lined processes appearing at the cell border increases concomitantly with hypertrophy of the lutein cell during the second week of pregnancy. Strands of particulate or grooved membrane emanate from the margin of larger gap junctions undergoing interiorization. Most large gap junctions are intimately associated with elements of the smooth endoplasmic reticulum. Spherical gap junctional profiles assume a deeper location in the lutein cell and may form concentric arrays by term while true surface gap junctions appear to fragment in the post-partum corpus luteum. The modifications observed are interpreted with respect to biogenesis of the gap junction and the hormonal control of lutein cell function.     

Linear gap and tight junctional assemblies between capillary endothelial cells in the eel rete mirabile

Background: Interendothelial tight junctions and gap junctions have been described in large blood vessels and in cultures of endothelium derived from large blood vessels. Transfer of microinjected smallmolecular weight tracers between adjacent endothelial cells also has been demonstrated indicating the presence of gap junctional interendothelial communication. Similar transfer of tracers is evident between microvessel endothelial cells in culture and in microvessels in situ. However, gap junctions have not been detectable by electron microscopy of intact capillary systems. This may be due to limited sampling available in diffuse capillary systems and a small area of overlap between adjacent endothelial membranes. Methods: Thin slices of the parallel, tightly packed capillary bed of the eel rete mirabile were cryofixed and prepared for conventional TEM by freeze substitution. Other samples were freeze-fractured and replicated for examination of endothelial junctional components. Results: A novel tight-gap junctional complex between rete capillary endothelial cells is described. In freeze-fracture replicas of the membrane P face, rows of gap junction subunits are flanked on either side by linear depressions representing grooves previously occupied by tight junctional strands that partition to the E face. In thin sections, the junctions appear in profile as short lengths of closely apposed membranes characteristic of gap junctions. Conclusions: The tight junctional components imply a barrier to paracellular transport across the capillary wall between the endothelial cells. The gap junctional component may provide a mechanism for communication between endothelial cells along the length of the vessel wall. © 1995 Wiley-Liss, Inc.     

Species differences in the synaptic membranes of the end bulb of held revealed with the freeze-fracture technique

Two morphological differences distinguish the membranes of the end bulb-spherical cell synapse in rats and mice from those in guinea pigs and chinchillas. First, in freeze-fracture replicas, the membranes of rat and mouse spherical cells lack perisynaptic aggregates which are present in the other species. Second, small gap junctions are present between the end bulb and spherical cell soma of rats and mice. These interspecies differences are not reflected in thin-sectioned material. This observation points out the difficulty in attempting to generalize about the significance of intramembrane specializations in synaptic membranes.     

A freeze-fracture study of intercellular junctions between various kinds of epithelial cells surrounding common endolymphatic space in the hearing organ of the chick

Intercellular junctions between various epithelial cells in the hearing organ (basilar papilla) of the chick were studied with the freeze-fracture technique. The effects of hypertonic solutions on the intercellular junctions were also examined. The basilar papilla of the chick is primarily composed of hair cells and supporting cells in the neuroepithelium, specialized columnar cells (TMC) which attach to the tectorial membrane, and light cells (LC) and dark cells (DC) in the tegmentum vasculosum. All of these epithelial cells surround a common endolymphatic space. The tight junctions between hair and supporting cells, and those between adjacent supporting cells in the neuroepithelium are 0.1–0.3 μm in depth and display the usual network of branching and anastomosing strands of shared intramembrane proteins. The tight junctions in the tegmentum vasculosum have the same structure as in the neuroepithelium. In contrast, the tight junctions between the TMCs are extremely well developed: They are 1–2 μm in depth. In freeze-fracture replicas, they appear as a fingerprint pattern of unbranched parallel particulate strands, running both parallel and perpendicular to the cell surface. After exposure to hypertonic solutions, all the epithelial cells are shrunken and intercellular spaces are expanded; all tight junctions, however, are intact. Thus, tight junctions in the basilar papillae are resistant to dissociation by hypertonic solutions. The usual zonulae occuludentes in the neuropithelium and tegmentum vasculosum are thought to prevent diffusion of endolymph through the intercellular spaces of epithelial cells. However, the tight junctions on the TMCs may function not only as a diffusion barrier, but also provide structural support to the cells anchoring the tectorial membrane which receives mechanical forces induced by the vibration of the basilar membrane. Extensive gap junctions are found between all the supporting cells (supporting cells in the neuroepithelium, TMCs, and LCs in the tegmentum vasculosum) surrounding the endolymphatic space.     

Freeze-fracture evidence for a novel restricting junction at the blood-brain barrier of the cuttlefish Sepia officinalis.

Summary The blood—brain barrier in the cuttlefishSepia officinalis has been studied with the freeze-fracture technique. Previous thin-section electron microscopy showed that a restricting junction is formed between perivascular glial processes in microvessels and venous vessels, and between pericytes in arterial vessels; the restriction appeared not to be a classicalzonula occludens or septate junction. In freeze-fracture replicas from brain optic and vertical lobe, endothelial cells, pericytes and perivascular glia could be recognized by their morphology and relation to the vascular lumen. In microvessels, endothelial and pericyte membranes showed sparse but uniform distribution of P-face intramembranous particles, with no particular particle aggregations. Perivascular glial membranes had a higher density of intramembranous particles but again no particle alignments characteristic of known restricting junctions were seen, although clusterings of intramembranous particles resembling gap junctions were present. In larger venous vessels, the perivascular glial layer showed a multilamellated organization, but again no arrays of intramembranous particles were detected, although this should be a favourable site for visualization of the restricting junctions. The walls of arterial vessels showed collagen deposits and cell processes with apparent intracellular myofilamentous profiles, but no intramembranous junctional particle arrays. It is concluded that the junctional zone observed in thin section electron microscopy is not associated with aligned aggregations of intramembranous particles detectable in freeze-fracture replicas, strengthening the evidence that this is a novel type of restricting junction.     

Permeability to lanthanum of blood testis barrier in human germinal aplasia

The permeability of Sertoli tight junctions to lanthanum administrated during fixation is demonstrated in biopsies of patients with partial germinal aplasia. In freeze-fracture replicas the number of fibrils is not significantly different from the data obtained in normal testis. Thus, in these pathological conditions junctional permeability is not related solely to the complexity of the network revealed by freeze-fracture.     

Junctional systems in the pineal gland of the Wistar rat (Ratus ratus). A freeze-fracture and thin section study

Intercellular junctions between neighbouring pinealocytes, glial cells, glial cells and pinealocytes as well as between nerve endings and parenchymal cells of the pineal gland of Wistar rats were investigated on freeze-fracture replicas and thin sections by transmission electron microscopy. Gap junctions, tight junctions and the annular gap junctions have been revealed. In addition, chemical synapses between nerve endings and pinealocytes have been observed.     

Cytoplasmic surface ultrastructures of cardiac gap junctions as revealed by quick-freeze, deep-etch replicas

Rapid-freeze, deep-etch, rotary-shadow replica studies were performed to examine the cytoplasmic surface membrane of the cardiac gap junctions of rats, mice, and guinea pigs. In quick-frozen fresh cardiac muscles, while the nonjunctional cytoplasmic surfaces were covered with filamentous materials, the cytoplasmic surface membrane continuous with freeze-fractured gap junction plaques were relatively free of such filaments and revealed particulate patterns. After brief rinsing in high K buffer, gap junction membranes showed granular substructures resembling a tiled surface made of round tiles of various sizes. After prolonged rinsing for more than 20 min, however, cytoplasmic surfaces of gap junctions became less particulate but rather smooth. The particulate substructures observed in the rapid-freeze deep-etch replicas may correspond to the fuzzy cytoplasmic layer in thin sections and serine protease sensitive peptide moiety in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reported in isolated cardiac gap junction pellets. These cytoplasmic components, which are absent in liver gap junctions, seem to be specific in cardiac and neural gap junctions and may be related to the large electrical current passed by these junctions.     

Fine structure of the afferent synapse and gap junctions on the sensory hair cell in the saccular macula of goldfish: A freeze-fracture study

Summary Membrane specializations at the active zone of the afferent synapse in the saccular macula of goldfish are described.Those of the presynaptic membrane consist of three to six elongated aggregates of intramembrane particles separated by particle-free furrows on the concave part of the P face and its complementary figure, that is, an alternate arrangement of elongated aggregates of pits and smooth ridges on the convex part of the E face. The size of the specialized area is about 0.5 m × 0.3 m.Vesicle fusion sites are situated at the margin of the particle-free furrow and ridge of the presynaptic active zone. Round pores about 30–50 nm in diameter are seen on the P face around the active zone. They are probably openings of the anastomosing tubules or coated pits.A focal aggregate of intramembrane particles is observed on the E face of the postsynaptic membrane apposing the presynaptic active zone. The P face of the postsynaptic active zone shows pits and particles.Small gap junctions are found between hair cells and adjacent supporting cells. They are frequently associated with desmosomes. The possible functional significance of these gap junctions is discussed.     

Freeze-fracture studies on the synapse between the type I hair cell and the calyceal terminal in the guinea-pig vestibular system

Summary The apposition between type I hair cells and the calyceal terminals of vestibular ganglion cell peripheral processes was studied in the vestibular epithelium of the guinea-pig, using thin-sectioned and freeze-fractured specimens. Chemical synaptic junctions were exceedingly rare in thinsectioned specimens, and were not seen in freeze-fracture replicas. Furthermore, no gap junctions were present between the hair cell and the calyx. There were, however, regions along the apposition where the membranes were closely apposed. At these regions, the hair cell was invaginated by cytoplasmic protrusions of the calyx and the plasmalemmata of the two cells were separated by only 6–7 nm. The number and conformation of the close appositions varied between different cells. In freeze-fracture replicas, the closely-apposed plasmalemmata of the hair cell and the calyx had no special distribution of intramembrane particles on either membrane leaflet. However, on the external membrane leaflet of the hair cell, a large patch of widely-spaced, large particles surrounded the regions of close apposition. The corresponding region of the plasmalemma of the calyx had no special distribution of particles on either membrane leaflet. The scarcity of chemical synaptic junctions, the absence of gap junctions between the cells and the unique arrangement of particles in the hair cell plasmalemma surrounding regions of close membrane apposition may indicate an unusual mode of synaptic transmission between the type I hair cell and the calyx.     

The association of gap junctions with large particles in the crypt epithelium of the rat small intestine

Intercellular junctions in the epithelium of the rat small intestine were studied by the freeze-fracture method. Gap junctions were found between columnar cells of the crypt far more frequently than between absorptive cells of the villus. In addition, in the crypt, large particles 11 to 13 nm in diameter were often associated with the gap junction which primarily consisted of usual gap junctional particles 8 to 9 nm in diameter. Both the usual and large particles possessed a central pit. The intestinal crypt is the site of epithelial cell proliferation and differentiation in the process of physiological cell renewal. The predominant presence of gap junctions in this region suggests that they play a certain role in the proliferation and differentiation of crypt columnar cells. Further studies are required to elucidate the nature of the large particles, a precursor of typical connexons and/or a functionally different gap junction.