Ultrastructure of the labyrinth in the rat full-term placenta

Summary The placental labyrinth of the chorioallantoic placenta of the rat was studied with transmission and scanning electron microscopy. Full-term placentas were investigated after perfusion fixation from the maternal and fetal circulation, including thin-sectioning and freeze-fracturing.The labyrinthal three-dimensional structure was found not to be trabecular but spongious lamellar. We propose a division of the lamellae into three groups—the first, second, and third order.The trophoblastic layers are described in detail, showing a cellular layer I and two syncytial layers II and III. Layer I is found to be fenestrated and highly permeable. Layers II and III are connected by extended gap junction areas. It is suggested that the gap junctions function as a molecular sieve and represent the limiting barrier in diaplacental transport of the choriollantoic placenta.Fetal capillaries are fenestrated and endothelial cells are connected by tight junctions.The functional significance of the morphologic findings is discussed.With the support of the Deutsche Forschungsgemeinschaft. Dedicated to Prof. Dr. Drs. h.c. W. Bargmann on his 70th birthday     

Regulation of the blood–biliary barrier: interaction between gap and tight junctions in hepatocytes

Hepatocytes tightly connect with each other by intercellular junctions to form liver cell plates. The junctions composed of gap, tight, and adherens junctions and desmosomes concentrate around bile canaliculi. In particular, tight junctions serve as a barrier to keep bile in bile canaliculi away from the blood circulation. Thus, it is very reasonable to call tight junctions of hepatocytes the blood–biliary barrier. On the other hand, gap junctions of hepatocytes are considered to enable ordered contraction of bile canaculi from centrizonal to periportal hepatocytes by their function of intercellular communication. Gap and tight junctions may thus play a crucial role in bile secretion, one of the most differentiated functions of the liver. In intrahepatic cholestasis, a common pathological condition of the liver, downregulation of gap and tight junctional functions is seen, which results in impaired intercellular communication and in leaky tight junctions. Although the changes in gap and tight junctions had been considered to be independent of each other, recent findings that the tight junction-associated proteins ZO-1 and occludin bind to connexins indicate the possibility of either coordinate or reciprocal regulation of macromolecular complexes containing gap- and tight-junction proteins. In this review, we introduce the interaction and regulation between gap and tight junctions of hepatocytes in vitro and discuss the regulatory mechanisms of the blood–biliary barrier to study the molecular pathogenesis of cholestasis.     

The gap junction family: structure, function and chemistry

Summary Gap junctions are aggregates of transmembranous channels which bypass the extracellular space by transporting messenger molecules and ions from one cytoplasmic source to an adjacent cytoplasmic interior. The channels join the plasma membranes of adjacent cells by bridging the extracellular space between them. Thereby, cellular compartments which were once considered to be individual units are, in actuality, interconnected by a system of pathways which form a functional cellular syncytium. The evolutionary importance of a generalized intercellular communication system can be appreciated when one considers the widespread prevalence of gap junctions within animals of all multicellular phyla, and within almost all tissues of vertebrates. Only a few population of cells such as skeletal muscle cells (which are fused to form functional syncytia) and circulating blood cells are not equipped with gap junctions. This paper provides a brief review of the diverse structural, molecular and functional aspects of gap junctions as revealed by current research.     

Electron microscopic studies on the myotomes of larval lamprey,Lampetra japonica

Myotomes of the caudal one-third of the body of 26-day-old larval lampreys, Lampetra japonica, were studied by electron microscopy. Each myotome consists of horizontally stacked muscle lamellae. The myotomes are covered laterally by a single layer of flattened cells called here “lateral cells,” and the other aspect is covered by an external lamina. The myotomes are mid-segmentally innervated. Each muscle lamella usually contains two single cortical layers of myofibrils along the dorsal and ventral sarcolemma with a nucleus and mitochondria interposed between two layers. Numerous peripheral couplings are observed with relatively less developed triads. There are no membrane specializations to connect adjacent muscle lamellae within a myotome. Intermyotomal junctions are, however, noted between tips of cytoplasmic processes of muscle lamellae of adjoining myotomes. They resemble tight or gap junctions. No myofibrils are present in these cytoplasmic processes. Myotendinous junctions, with “terminal couplings” (Nakao, 1975), are seen under development at the myoseptal ends of muscle lamellae. Lateral cells contain only ordinary organelles and no special structures such as myofibrils are found in the cytoplasm. They are connected to each other and to muscle lamellae by primitive desmosomes. They generally have no external lamina investment.     

Perineurium of sciatic nerve in normal and diabetic rodents: freeze-fracture study of intercellular junctional complexes

Summary A comparative study has been carried out using the freeze-fracture technique on the perineurium of the sciatic nerve from normal and diabetic mice (C57B1/Ks, BALB/c and CD₁ strains) and rats of various ages. The replicas showed that tight junctions connected perineurial cells both within the same cell layer (zonulae occludentes) and between adjacent layers (maculae occludentes). In neonates, a number of zonulae occludentes were characterized by short, incomplete or fragmented ridges at various intervals from each other; in adults, tight junctions appeared as mature networks of interconnected, branching and/or anastomosing strands. Zonulae occludentes of diabetic mice also exhibited frequent interruption of the strands and reduction in the branching of strands. Gap junctions occurred in both zonulae and maculae occludentes of normal and diabetic rats at all ages. In the C57B1/Ks strain such junctions occurred more frequently in zonulae occludentes of diabetic animals. It is suggested that perineurial cells are coupled by gap junctions to allow fast transfer of ions and small-sized molecules across the layers; under pathological conditions, such as diabetes, the increase in cell-to-cell signalling may be important in controlling the abnormal metabolic situation.     

An experimental analysis of interlamellar tight junctions in amphibian and mammalian C.N.S. myelin

Summary The distribution of interlamellar tight junctions was examined in myelin sheaths ofXenopus tadpole optic nerve and rabbit epiretinal tissue fixed with aldehydes, postfixed with osmium ferrocyanide and embedded in a water-soluble medium, Durcupan. Intramyelinic zonulae occludentes were clearly formed by fusion of adjacent intraperipd lines which corresponded to the external leaflets of oligodendrocytes. These occurred in register with other tight junctions present within successive lamellae and appeared as a series of radial lines extending either partially or totally across the thickness of the myelin sheath. This distribution of zonulae occludentes corresponded with that of tight junctional particle strands observed in freeze-fracture replicas.Analysis of intramyelinic vacuolation induced by hexachlorophene (HCP) intoxication indicated that lamellar splitting was frequently limited by the tight junctions. The intramyelinic zonulae occludentes also restricted the diffusion of colloidal lanthanum which had penetrated the myelin intraperiod gap followingin vivo perineural injection. The results of this study provide evidence favouring a correspondence between interlamellar tight junctions and the radial component of myelin described earlier by other investigators. Furthermore, observations of swollen myelin sheaths, resulting from HCP intoxication, suggest that these junctions may play a major role in maintaining myelin sheath integrity and limiting the extent of breakdown during certain pathological conditions.     

Membrane specializations and their relation to HRP transport in the medial habenular nucleus of the rat

Summary In the medial habenular nucleus of the rat, ependymal and endothelial membrane specializations were studied with TEM and freeze-fracturing. They comprise ependymal adherent junctions — not manifest in freeze-fracture replicas-, gap junctions, and membrane-associated orthogonal particle complexes (assemblies) — not identifiable in thin-sectioned material. Ependymal tight junctions being absent, no brain-liquor barrier exists. The capillary endothelium is provided with tight junctions only.Intraventricularly injected HRP was transported in large amounts through the ependyma, mainly through the intercellular spaces and additionally by way of massive pinocytosis through the cytoplasm of particular ependymal cells only, and finally through the parenchymal intercellular compartments towards habenular capillaries. Following intravenous injection of HRP, considerable transport of the enzyme took place by means of transendothelial pinocytosis, followed by some pinocytotic transport through diverse parenchymal elements and markedly profuse incorporation and lysis within pericytes. The habenular blood-brain barrier appeared to be considerably leaky with respect to HRP.     

The incidence and size of gap junctions between the bone cells in rat calvaria

Polyclonal antisera to synthetic peptides matching sequences on the cytoplasmic regions of connexin-43, a gap junction protein first identified in rat heart, have been used to immunolabel gap junctions in the calvarial bone, maintained intact as in vivo, of 1- to 2-week-old rats. The specimens were examined in reflection and fluorescence modes by scanning laser confocal microscopy, and the numbers of gap junctions and their sizes estimated. The mean number of connexin-43 immunolabelled junctions per osteoblast (n=65) was 15.3 (SD ± 4.5). The mean length of 227 junctions, selected for the sharpness of the image of the fluorescent spot, was 0.67 m (SD ± 0.18; range 0.37–1.29 m) and their mean area 0.26 m² (SD ± 0.145; range 0.075–0.93 m²); these probably fell within the upper half of the total size range. Gap junctions were detected between preosteoblasts, osteoblasts, osteocytes and chondrocytes, and between these juxtaposed cell types. In addition, connexin-43 immuno-labelled junctions were found between some osteoclasts and overlying mononuclear cells at active sites of resorption.     

Tight junction contact events and temporary gap junctions in the sciatic nerve fibres of the chicken during Wallerian degeneration and subsequent regeneration

Summary Tight and gap junctions are described on the basis of freeze-fractures in normal chicken sciatic nerves as well as during Wallerian degeneration and subsequent regeneration. 1. Small calibre nerve fibres display a fairly continuous tight junction contact zone in the membranes of the mesaxons, paranodal loops and Schmidt-Lanterman incisures. Large fibres with more than 40 lamellae have only focal tight junction contacts in the mesaxonal membranes. 2. With the onset of Wallerian degeneration (days 2–4 post-crush, distal stump) myelinic tight junctions become arranged as maculae composed of one circular or several polygonally oriented strands that are criss-crossed by other tight junctional strands. These maculae are subsequently found in the membranes of cytoplasmic vacuoles of the Schwann cells, indicating an endocytotic mode of uptake. Tight junctions are not found between the 5th and 6th day after crush. 3. During the proliferation phase of the Schwann cells and the arrangement of these cells into Büngner cell bands (2 to 8 days post-crush) gap junctions appear between the Schwann cells of the bands. These junctions then disappear with the onset of remyelination (8 days post-crush). 4. With the onset of remyelination (from the 8th day onwards) short focal tight junctions appear in the membranes of the outer mesaxons. Shortly thereafter, when the sheaths possess 4 to 8 lamellae, tight junctions also appear in the membranes of the inner mesaxons, the paranodal loops and the cytoplasmic inclusions. The characteristic differences of tight junction elaboration in small versus large nerve fibres are re-established after three months of regeneration.The elaborated tight junctions in small and early remyelinating fibres point to a specific function; in small fibres (versus large fibres) the tight junctions might effect a separation of the intramyelinic extracellular space as a single compartment. The tight junction contacts in early remyelinating fibres support the hypothesis that myelin growth occurs within the myelin spiral and not by a free rotation and elongation of the Schwann cell tongues. It is assumed that the gap junctions between the Schwann cells contribute to the co-ordination of the Schwann cell band formation, which is involved in the guidance of sprouting axons.     

Intercellular junctions between specialized ependymal cells in the subcommissural organ of the rat

Summary The permeability of intercellular junctions in specialized ependymal cells in the rat subcommissural organ (SCO) has been studied ultrastructurally by freeze-fracturing and tracer experiments with horseradish peroxidase (HRP). In addition to normal smooth membrane, areas which could be classified as a leaky tight junction are found within the ependymal junctional region. This consists of only one or two relatively continuous strands but with interruptions in the apical portion. Some strands are perpendicular to the apical membrane surface and often form hairpin-like bends in the basal portion of the junction. The junctional region also shows areas with no strands but only a rippled membrane structure which may be equivalent to very close appositions without fusion of adjacent ependymal cell membranes. The relative proportions of normal smooth membrane, strands and rippled structure in the junctional region is approximately 346 including two parts overlapping of the strands and rippled structure. Intraventricularly infused HRP passes through many junctions but is occasionally stopped, leaving unstained intercellular spaces of various lengths between membrane fusions of tight junctions. Even when it is stopped, the intercellular space below the junction is densely stained by the enzyme. Orthogonal arrays of intramembrane particles are found to be distributed on the basal and lateral cell membranes below the junctional region in the SCO ependyma.     

Structure of the musculature of the chicken small intestine

Summary The small intestine of the chicken was studied by light and electron microscopy. The musculature, measuring about 180 m in thickness in the distended intestine, consists of four layers (outer longitudinal, outer circular, inner circular and inner longitudinal) which are directly apposed to one another. There is no layer of connective tissue equivalent to the submucosa of mammalian intestine, and the intestinal glands lie close to the inner longitudinal muscle. Mucosal folds are not formed during isotonic contraction of the intestine. The muscle cells of the chicken small intestine are characterized by large, numerous and sharply outlined dense bodies, by the presence of an extremely thin basal lamina, by prominent dense bands at the cell surface but relatively few intermediate junctions. There are many areas of direct apposition between cell membranes of adjacent cells and little collagen between the muscle cells. The four muscle layers have each distinctive structural features. Gap junctions between muscle cells occur only in the outer circular layer. The outer circular and outer longitudinal layers are closely apposed and numerous junctions of the adherens type link cells of the two layers. Intramuscular blood capillaries are rare and are found virtually only in the outer circular layer; their endothelial cells are joined by tight junctions. In the outer circular layer (but not in the other layers) there are two further cell types, fibroblasts and interstitial cells, which can be clearly distinguished from one another. The latter cells are intimately related to nerve bundles and are connected by gap junctions to some muscle cells.     

Morphological indications for considerable diffuse reabsorption of cerebrospinal fluid in spinal meninges particularly in the areas of meningeal funnels

Transmission and scanning electron microscopical observations in the rat indicate a considerable capacity of the spinal meninges to reabsorb cerebrospinal fluid. The density of blood vessels and lymphatics in the duramater is extremely high, particularly in the areas of meningeal funnels and spinal nerve root sleeves. Arterioles with closely related unmyelinated nerve fibres, many fenestrated capillaries and venules predetermine these areas as sites where absorption processes could take place. At certain sites of the meningeal angle region, the arachnoid membrane, mostly multilayered, is reduced to only three or four layers. Intercellular discontinuities and cytoplasmic fenestrations occurring in the arachnoid lining cell layer result in direct communications between the subarachnoid space and cisterns of the arachnoid reticular layer. These cisterns are partly fluid-filled, partly occupied by a net of collagen fibre bundles. Some cisterns harbour macrophages that often project filiform processes through the lining cell layer into the subarachnoid space, contacting cerebrospinal fluid. Desmosomes and gap junctions are present in all layers of the arachnoid. However, tight junctions and the continuous electrondense intercellular gap, known to occur normally within the arachnoid barrier layer, were not seen in many sites of the meningeal angle region. Numerous arachnoid cells display a high degree of vesiculation. Cationized ferritin, introduced in vivo into the rat subarachnoid space, passes inter- and intracellularly from the cerebrospinal fluid compartment through the arachnoid membrane, reaching durai blood vessels and lymphatics. Tracer could be visualized both in the cytoplasm of the endothelium and on the luminal surface of the cells. Tracer also passed through pial cell layers into pial vessels, through leptomeningeal sheaths into vessels crossing the subarachnoid space, into the connective tissue compartment and into vessels of spinal dorsal root ganglia. In the angle region, a particularly large number of macrophages can be found on the surface of leptomeninges, within the arachnoid reticular layers, and in close relation to dural and epidural capillaries, venules and lymphatics. Their possible role in the process of cerebrospinal fluid reabsorption is discussed.     

Characterization of intercellular junctions in the caudal portion of the developing neural tube of the chick embryo

The types of intercellular junctions present within caudal levels of the chick neural tube (i.e., future lower thoracic and lumbosacral regions of the spinal cord) were determined by freeze-fracture of stage 14 to 16 embryos. Two levels of the developing neural tube were examined: the region of the neurulation overlap zone–consisting of primary neural tube dorsally and secondary neural tube ventrally–and the portion of the primary neural tube located just cranial to the overlapping region. Gap junctions were the most numerous type of intercellular junction present within the primary neural tube. These junctions were located primarily in juxtaluminal areas, near the apices of neuroepithelial cells, and sometimes also at the bases of these same cells. In addition, focal, poorly defined tight junctions occasionally occupied juxtaluminal regions of the primary neural tube. The medullary cord (i.e., the immediate precursor of the secondary neural tube) and secondary neural tube contained gap junctions exclusively. Gap junctions were first found in these areas at the lateral borders of the medullary cord, concomitant with formation of this structure, and then at the interface between the elongated, peripheral cells of the cord and the irregularly shaped and loosely arranged central cells of this structure. Finally, gap junctions were distributed radially around secondary lumina formed by cavitation. The precise spatial and temporal correlation between the appearance of gap junctions and the specific changes occurring in cellular morphology and arrangement during secondary neurulation strongly suggest that gap junctions may have a role in coordinating cellular activities during formation of both the medullary cord and the secondary neural tube.     

Fine-structural investigation of rat brain microvascular endothelial cells: Tight junctions and vesicular structures in freshly isolated and cultured preparations

Summary A comparison was made between endothelial cells in freshly-isolated rat brain microvessels, and following culture of the cells for 1–10 days during growth to confluence. Attention focused on tight junctions and vesicular structures, as seen in thin sections and freeze-fracture replicas. Freshly-isolated vessels had an abnormal appearance, with a profusion of luminal microvillar processes, and extensive cytoplasmic vacuolation. There were numerous vesicular profiles, reaching a density of 60 m^–2, and with a large proportion open to the surface, as shown by labelling with cationized ferritin at 4 °C for 5 min. Junctional zones were relatively loosely organized, with evidence for some cell: cell separation, as well as some residual tight junctional sites withinzonula adhaerens junctions. In freeze-fracture replicas, Junctional strands showed segments of tightly packed intramembrane particles, generally on the P face. After 1 day in culture, the cells appeared more normal, with no vacuolation or luminal processes. Vesicles were still numerous, some associated with junctional zones, while tight junctions were relatively sparse; freeze-fracture showed some incomplete tight junctional strands, with some of the intramembrane particles fracturing onto the E face. The double offset fibrillar nature of the strands could occasionally be seen. Cells cultured for 4 and 10 days showed a progressive increase in the completeness of the junctional zone, with more tight junctional contacts within the length of theadhaerens junction, and an aggregation of microfilaments in the underlying cytoplasm. The number of vesicular profiles declined, and they were progressively excluded from the junctional zone. These observations have relevance for studies on the physiology of the brain endotheliumin vitro, and for comparisons with thein vivo condition.     

Occluding Junctions in an Epithelioid Mesothelioma: A Freeze-Fracture Study

The presence of a well-developed tight junction system in a case of diffuse, papillary, malignant mesothelioma of the peritoneum is documented using the freeze-fracture methodology. The mean number of strands within the tight junction network was determined to be 3.83 i 0.16. In addition, the tight junctions were specifically located in regions of the plasma membrane adjacent to recognizable microvilli. Gap junctions and desmosomes were found in close association with the occluding complexes; however, they were also seen in areas of the plasma membrane quite distinct from those that exhibit tight junction differentiation. The possible significance of the presence of tight junctions in neoplastic epithelial cells is also discussed.     

Tight junctions in the ependyma of the spinal cord of the urodele Pleurodeles waltlii

Summary The ependymal junction pattern in the spinal cord of postmetamorphic ribbed newts has been studied, using transmission electron microscopy of ultrathin sections of normal animals and of animals perfused through the IVth ventricle with lanthanum. Contrary to what has been observed in mammalian CNS, the ependyma of the urodelan spinal cord is furnished with tight junctions that seal the luminal border of the terminal bars. These occludens junctions are made up of two to seven punctate fusions of the plasma membranes. Lanthanum tracer remains restricted inside the lumen of the central canal, being stopped at the first punctate fusion on its way through the intercellular clefts. Beyond this point, the extracellular space contains no tracer material. Besides tight junctions, intermediate, desmosomal and gap junctions are also present. Gap junctions and desmosomes are not present in CSF-contacting neurons. It is suggested that ependyma with occluding junctions (special ependyma) overlay the regions of the CNS where the ependymal cells significantly modify the composition of both intercellular and cerebrospinal fluids, through secretory, transporting and permeability control activities.Dedicated to Dr. Jean Emmanuel Gruner     

A freeze-fracture study of the guinea pig yolk sac epithelium

The yolk sac epithelium functions in endocytic absorption of macromolecules from the uterine lumen and in maintaining permeability barriers between the maternal (uterine) compartment and underlying fetal compartments. In this study, cell membranes and intercellular junctions of the guinea pig yolk sac were examined using the freeze-fracture technique. Intramembranous particle (IMP) distribution and size were examined in microvilli, intermicrovillous membrane, and endocytic pits. IMPs on the P-fracture face were not evenly distributed within these membrane domains and the IMPs were of several sizes. In addition, short filamentous strands sometimes bridged between the cytoplasm and the E-face of endocytic pit membranes. These results indicated that the regions of yolk sac cell membrane involved in endocytosis did not have unusual numbers or distributions of IMPs compared to other membrane regions. The intercellular junctions between endoderm cells consisted of zonulae occludentes, gap junctions, and desmosomes. The zonula occludens usually consisted of four or five interconnected strands or ridges, and were elaborately developed where three cells abutted one another. Gap junctions were infrequent, small, and often associated with the zonula occludens. Desmosomes were observed both as typical macular structures and as more extensive, elongated structures. These results confirm previous thinsection studies of yolk sac indicating the presence of tight junctions. However, the extent of development of tight junctions does not correlate with the reported absence of a potential difference across yolk sac epithelium. The presence of gap junctions does not confirm results of previous freeze-fracture studie sof other yolk sacs.     

Intercellular junctions in upper airway submucosal glands of the rat: a tracer and freeze fracture study

The structure of intercellular tight junctions of rat airway submucosal glands was examined by freeze fracture techniques and their permeability assessed by the use of colloidal lanthanum. The submucosal glands were organized into three distinct regions: a) serous tubules and b) mucous tubules lined, respectively, by serous and mucous cells, and c) ducts lined by cuboidal epithelial cells, containing few secretory granules, and some ciliated cells. The mean number of parallel fibrils constituting the tight junctions between serous cells was 3.6 +/- 0.4, which was significantly smaller than those between any of the other cell types. Colloidal lanthanum permeated the tight junctions between serous cells up to the level of the acinar lumen. There was a progressive increase in the mean number of parallel fibrils of tight junctions between mucous (5.1 +/- 0.6), ductal (5.4 +/- 0.5), and ciliated cells (8.5 +/- 0.7); none of these junctions was permeated by colloidal lanthanum. These results imply that tight junctions between serous cells are more permeable to small water-soluble solutes than those present in the more proximal portions of the gland. Gap junctions were observed between serous cells and between mucous cells, suggesting that these secretory cells may be electotronically and metabolically coupled.     

Freeze-fracture study of the perineurium around frog dorsal root ganglia

Summary The perineurium around frog dorsal root ganglia consists of layers of flattened cells separated by extracellular connective tissue elements. The number of layers is smaller than that in the perineurium around adjacent peripheral nerves, and some of the layers are discontinuous, but in both cases, cells in the same layer overlap and form tight junctions with each other, sometimes accompanied by desmosomes or gap junctions. In freeze-fracture replicas the tight junctions between perineurial cells around peripheral nerves consist of 13–91 strands (mean: 38). Some of these are parallel to the cell borders and some are oblique, forming elaborate meshworks. The overall width of each junction averages 12 (m. In contrast, the tight junctions between perineurial cells around ganglia are much narrower, averaging 2 m in width, and they consist of only 1–14 strands (mean: 6) with few anastomoses and many free ends. These structural differences provide a morphological basis for a less complete diffusion barrier around dorsal root ganglia.     

Tight junctions in neurological diseases

Tight junction, one of the type of cell-cell junctions, controls the paracellular permeability across the lateral intercellular space and maintains the cell polarity. Tight junctions consist of transmembrane proteins: members of tight junction-associated MARVEL protein (TAMP) family, claudins and junctional adhesion molecules (JAMs), and various cytoplasmic proteins that are necessary for the correct organization of the integral membrane components of the junction. Alterations in expression or localization of proteins of tight junctions have been described in several neurological disorders including multiple sclerosis, stroke, Alzheimer's disease, Parkinson's disease and epilepsy. In this review, we summarize the most recent data on components of tight junctions and focus on the implication of tight junction dysfunction in neurological diseases.