Studies on perineural junctional complexes and the sites of uptake of microperoxidase and lanthanum in the cockroach central nervous system

The perineurial junctional complexes in the nerve cord of Periplaneta americana have been shown to consist of septate desmosomes, extensive gap junctions and relatively limited regions of tight junctions. Microperoxidase (M.W. 1,900) undergoes limited intercellular penetration into the septate desmosomes. Lanthanum penetrates both the septate desmosomes and gap junctions. It is concluded that the restricted access of these substances to the underlying extracellular spaces results from the presence of the perineurial tight junctions. These results contrast with those for small peripheral nerves, which lack equivalent junctional complexes, and in which the extracellular spaces are found to be accessible to externally applied lanthanum. The results are discussed in relation to current concepts of the insect blood-brain barrier.     

A vertebrate-like blood--brain barrier, with intraganglionic blood channels and occluding junctions, in the scorpion

India ink and ionic lanthanum injections have revealed that the central nervous system (CNS) of the scorpion possesses a highly vascularized cephalothoracic ganglionic mass. It, together with other abdominal ganglia which form a ventral nerve cord, are all ensheathed by an outer layer of modified glial, or perineurial, cells. These cells resemble those which line the blood channels permeating the CNS, in exhibiting both inverted gap and tight junctions. Although the latter show close or fused membrane appositions, lanthanum appears to penetrate past a number, but not all, of them. Freeze-fracturing reveals that these junctions are composed of E-face particles aligned into a network of rows, or ridges, which are frequently discontinuous, especially near the periphery of the perineurium. This produces a somewhat 'leaky' system but occlusion to tracers occurs ultimately, for in the CNS none can be found beyond the perineurium. The existence of this perineurial blood-brain barrier is also demonstrable electrophysiologically where cations such as Mg2+ are unable to penetrate beyond the perineurial layer although they can, it seems, leak in via the blood vascular system. Relative differences in tightness between the perineurium and the cells lining the blood channels may be attributed to differences in the relative number of discontinuous ridges. This is borne out by the observation that the peripheral nervous system has a highly attenuated perineurium with many fewer junctions, and some of these nerves tend to be leaky with respect to tracer penetration. In fixed material the junctional ridges may fracture on to the E-face or partly on both the EF and PF, while in unfixed tissue they are usually found on the PF. In both cases they exhibit complementary grooves that are coincident with the ridges across membrane transitions; in such cases the cell membranes are fused with concomitant obliteration of the intercellular space. These tight junctions, often closely associated with EF gap junctional particle aggregates which may be very loosely clustered, appear to form the basis of the observed blood-brain barrier in the scorpion CNS.     

Biological implications of gap junction structure,distribution and composition: A review

Traditionally, all gap junctions have been considered to be identical in structure and function throughout the animal kingdom. Functions ascribed to these membrane specializations have been fundamental and have not been thought to differ significantly with respect to their mechanism of action. More recent studies support the view, however, that structural and compositional diversity may reflect significant functional differences between gap junctions in different classes of tissue but no clear and definitive patterns have yet emerged. This review does not attempt to comprehensively analyze the totality of the vast gap junction and coupling literature but focuses instead upon those recent observations which raise new questions related to the biological activities of gap junctions in different tissues.     

Arthropod fine structure: Towards an understanding of the intricacies of intercellular junctions

The technical advances in electron microscopical methodology over the past few decades have made it possible to come to an understanding of the nature of intracellualr junctions Tracer and freeze-fracture studies on intact structures have revealed the intracellular and intermembranous features of junctions in arthropods which exhibit interesting differences from those of vertebrates The well-nigh ubtiquitous gap junctions of arthropods exhibit interesting differences from those of vertebrates. The well-nigh ubiquitous gap junctions of arthropods possess unusual fracturing characteristics which makes it possible to follow readily their development, uncoupling and turnover. Tight junctions, originally thought not to exist in the invertebrates, are now unequivocally established to be present in tissues of some arthropods. Septate junctions, unique to the invertebrates and very widespread within that kingdom, exhibit a variety of substructure in different tissues. Desmosomes also exist in a range of forms. Rapid freezing without prior fixation of tissues has elucidated certain other features of these structures, while preliminary work on isolated junctions and antibodies raised to them permits their immunocytochemical EM analysis.     

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell–cell and cell–matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.     

Biochemical and biophysical analysis of cell-to-cell channels and regulation of gap junctional permeability

Gap junctional communication is a universal property of metazoan animals. Biochemical, immunological, molecular biological, ultrastructural, biophysical and physiological studies of gap junctions have permitted increasingly detailed modelling of gap junctional structure and function. In spite of this progress the questions to be addressed are whether the channel is a mixed oligomer and the stoichiometry for each tissue is fixed. Also the extent of homology among gap junction proteins in different tissues and their possible regulatory function have to be clarified. As long as the different channels are not cloned and expressed, the ultrastructural correlates of the physiological concepts such as channel gating, selectivity and regulation, as well as assembly and disassembly cannot be determined. The genetic approach is in full progress. The observed differences between gap junction proteins from different tissues and the multiplicity of subunits in even one channel implies a functional specialization for gap junctions. Correlative studies on the molecular and cellular level should help to clarify the physiological meaning of intercellular communication by gap junctions.     

MYONEURAL JUNCTIONS OF TWO ULTRASTRUCTURALLY DISTINCT TYPES IN EARTHWORM BODY WALL MUSCLE

The longitudinal muscle of the earthworm body wall is innervated by nerve bundles containing axons of two types which form two corresponding types of myoneural junction with the muscle fibers Type I junctions resemble cholinergic neuromuscular junctions of vertebrate skeletal muscle and are characterized by three features: (a) The nerve terminals contain large numbers of spherical, clear, ~500 A vesicles plus a small number of larger dense-cored vesicles (b) The junctional gap is relatively wide (~900 A), and it contains a basement membrane-like material, (c) The postjunctional membrane, although not folded, displays prominent specializations on both its external and internal surfaces The cytoplasmic surface is covered by a dense matrix ~200 A thick which appears to be the site of insertion of fine obliquely oriented cytoplasmic filaments The external surface exhibits rows of projections ~200 A long whose bases consist of hexagonally arrayed granules seated in the outer dense layer of the plasma membrane The concentration of these hexagonally disposed elements corresponds to the estimated concentration of both receptor sites and acetylcholinesterase sites at cholinergic junctions elsewhere. Type II junctions resemble the adrenergic junctions in vertebrate smooth muscle and exhibit the following structural characteristics: (a) The nerve fibers contain predominantly dense-cored vesicles ~1000 A in diameter (b) The junctional gap is relatively narrow (~150 A) and contains no basement membrane-like material, (c) Postjunctional membrane specialization is minimal. It is proposed that the structural differences between the two types of myoneural junction reflect differences in the respective transmitters and corresponding differences in the mechanisms of transmitter action and/or inactivation.     

Intercellular junctions in the hepatopancreas of the lobster Nephrops norvegicus

The hepatopancreas of the lobster has recently been found to be a rich source of material from which to isolate arthopod gap junctions biochemically (Finbow et al., 1983a; 1984). It has therefore been studied here to assess the features of these intercellular junctions and any others that may be present, in vivo. The tissue consists of columnar epithelial cells which possess apical microvilli and basal infoldings. In thin sections the lateral borders of these cells are characterized by desmosomes and smooth septate junctions as well as by gap junctions. The desmosomes exhibit no apparent freeze fracture profile but the septate junctions display parallel rows of ridges or aligned intramembranous particles (IMPs) with complementary grooves on the other membrane half; these IMPs shift in their preferential fracturing plane depending on whether the tissue has first been fixed, always remaining on the EF if unfixed. The IMPs or connexons, of which the gap junctions are composed, fracture onto the E face, leaving complementary pits on the P face, regardless of whether the tissue is fixed or not. At the base of the pancreatic cells, the lateral borders are thrown into interdigitating folds which display endocytotic profiles and possible internalization of junction-bearing membranes. This phenomenon, which is readily visualized both after tracer incubation and in replicas, may represent junctional degradation relating to membrane turnover.     

Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis

Direct immunogold labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a "control" tissue to identify preparation and labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during labeling, and several factors limiting quantitative analysis.     

Change of form of septate and gap junctions during development of the insect midgut

In insects, smooth septate junctions join cells derived from the embryonic midgut, and pleated septate junctions are found in all other tissues. Relatively little is known about either type of septate junction or the relationship between them, but they have been treated as two different junctions in the literature. The gap junctions which are associated with these septate junctions also differ. Crystalline gap junctions are found in the midgut, associated with smooth septate junctions, and irregular gap junctions are found in tissues where pleated septate junctions are located. We have examined the development of smooth septate junctions and crystalline gap junctions and the relationship between them, by studying the embryogenesis of the midgut in Manduca sexta (tobacco hornworm). At 56 hr of development (hatching is at 104 hr) pleated septate junctions and irregular gap junctions joined the midgut epithelial cells. At 65 hr, the septate junctions had disappeared, but gap junctions persisted. At 70 hr, smooth septate junctions had replaced the earlier pleated septate junctions and gap junctions associated with these smooth septate junctions were often of the crystalline form. In later embryos, the smooth septate junctions matured and enlarged, while all gap junctions became crystalline in form.     

The ultrastructure of the rat primary decidual zone

The rat primary decidual zone (PDZ) is a transitory, avascular region of transformed fibroblasts surrounding the implanting embryo. Tracer studies have indicated that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight of the tracer. To clarify the morphological basis of the permeability barrier, we have studied the ultrastructure of the PDZ with particular emphasis on the intercellular features and cellular junctions. The cells of the PDZ were large and tightly packed; their apposed membranes showed extensive interdigitations in some regions, but elsewhere they were relatively straight. Tight junctions, gap junctions, and desmosomelike junctions were observed between decidual cells. The tight junctions usually consisted of one or two points of membrane fusion, and they were oriented both parallel and perpendicular to the long axis of the PDZ. These junctions were frequently associated with gap junctions. Scattered pockets of dilated extracellular space between decidual cells contained collagen fibrils and an amorphous, dense material. These extracellular components were also sequestered by the decidual cells in deep invaginations of the cell surface that were continuous with the extracellular space. Decidual cells also exhibited flangelike processes that penetrated the basal laminae of the adjacent epithelium and capillary endothelium. Our present observations indicate that decidual cells are connected by tight junctions, and a previous study demonstrated that macromolecules up to 40 kDa readily cross the PDZ; hence, the tight junctions appear to be discontinuous. We suggest that the structures restricting the movement of large macromolecules (66 kDa and larger) across the PDZ from blood vessels to the embryo may include discontinuous tight junctions, membrane interdigitations, and amorphous intercellular material.     

Junctional types in the tissues of an onychophoran: the apparent lack of gap and tight junctions in Peripatus

The Onychophora are a rare group of primitive invertebrates, relatively little investigated. Tissues from a range of their digestive, secretory and excretory organs have been examined to establish the features of their intercellular junctions. Glutaraldehyde-fixed cells from the midgut and rectum, as well as the renal organ, mucous gland, salivary gland, epidermis, CNS and testis from specimens of Peripatus acacioi, have been studied by thin section and freeze-fracture electron microscopy. Adjacent cells in the epithelia of all these tissues are joined by apical zonulae adhaerentes, associated with a thick band of cytoskeletal fibrils. These are followed by regular intercellular junctional clefts, which, in thin sections, have the dense, relatively unstriated, appearance of smooth septate junctions (SSJ). However, freeze-fracture reveals that only the midgut has what appear to be characteristic SSJs with parallel alignments of closely-packed rows of intramembranous particles (IMPs); these IMPs are much lower in profile than is common in such junctions elsewhere. The mucous gland, testis, rectal and renal tissues exhibit, after freeze-fracture, the characteristic features of pleated septate junctions (PSJ) with undulating rows of aligned but separated junctional particles. Suggestions of tricellular septate junctions are found in replicas at the interfaces between 3 cells. In addition, renal tissues exhibit scalariform junctions in the basal regions of their cells. Between these basal scalariform and apical septate junctions, other junctions with reduced intercellular clefts are observed in these renal tissues as well as the rectum, but these appear not to be gap junctions. Such have not been unequivocally observed in any of the tissues studied from this primitive organism; the same is true of tight junctions.     

Localization and distribution of gap junctions in normal and cardiomyopathic hamster heart

Gap junctions in mammalian heart function to provide low-resistance channels between adjacent cells for passage of ions and small molecules. It is clear that the almost unrestricted passage of ions between cells, ionic coupling, is required for coordinate and synchronous contraction. This knowledge of gap junction function has made it important to study their properties in normal and abnormal tissues. In the present study, we analyzed gap junction distribution in normal and cardiomyopathic heart tissue utilizing immunofluorescent and electron microscopy techniques. Frozen, unfixed sections of age-matched normal and cardiomyopathic cardiac tissues were immunofiuorescently stained using an antibody directed against a specific peptide sequence of the connexin-43 gap junction protein. These studies revealed a characteristic punctate staining pattern for the intercalated discs in normal tissues. Some of the intercalated discs in cardiomyopathic hearts appeared to stain normally; however, others stained diffusely. The pixel intensity distribution of the confocal images demonstrated a marked difference of up to 90% increase in the number of pixels in cardiomyopathic myocardium (CM), yet the pixel intensity of gap junctions had a decrease of approximately 60%. This suggests the possibility that connexin-43 is present in CM cells in significant quantity; however, it does not become localized on the membranes as in normal cells. Electron-microscopic findings corroborate these observations on CM cells by showing an irregular distribution of intercalated discs relatively smaller in size with abnormal orientation and distribution. © 1994 Wiley-Liss, Inc.     

Gap junctions in several tissues share antigenic determinants with liver gap junctions.

Using affinity-purified antibodies against mouse liver gap junction protein (26 K), discrete fluorescent spots were seen by indirect immunofluorescence labelling on apposed membranes of contiguous cells in several mouse and rat tissues: pancreas (exocrine part), kidney, small intestine (epithelium and circular smooth muscle), Fallopian tube, endometrium, and myometrium of delivering rats. No reaction was seen on sections of myocardium, ovaries and lens. Specific labelling of gap junction plaques was demonstrated by immunoelectron microscopy on ultrathin frozen sections through liver and the exocrine part of pancreas after treatment with gold protein A. Weak immunoreactivity was found on the endocrine part of the pancreas (i.e., Langerhans islets) after glibenclamide treatment of mice and rats, which causes an increase of insulin secretion and of the size as well as the number of gap junction plaques in cells of Langerhans islets. Furthermore, the affinity purified anti-liver 26 K antibodies were shown by immunoblot to react with proteins of similar mol. wt. in pancreas and kidney membranes. Taken together these results suggest that gap junctions from several, morphogenetically different tissues have specific antigenic sites in common. The different extent of specific immunoreactivity of anti-liver 26 K antibodies with different tissues is likely due to differences in size and number of gap junctions although structural differences cannot be excluded.     

A FINE STRUCTURAL ANALYSIS OF INTERCELLULAR JUNCTIONS IN THE MOUSE LIVER

Zonulae occludentes and gap junctions were examined both in the intact mouse liver and in a junction-rich membrane fraction from homogenized mouse liver. These preparations were visualized with the techniques of uranyl acetate staining en bloc, staining with colloidal lanthanum, negative staining with phosphotungstate, and freeze-cleaving. The zonula occludens is arranged as a meshwork of branching and anastomosing threadlike contacts sealing the lumen of the bile canaliculus from the liver intercellular space. The gap junction is characterized in section by a 20 A gap between the apposed junctional membrane outer leaflets, and permeation of this space with lanthanum or phosphotungstate reveals a polygonal lattice of subunits with a center-to-center spacing of 90–100 A. Freeze-cleaved gap junctions show a similar lattice. Extraction of junction-rich fractions with 60% aqueous acetone results in a disappearance of the 20 A gap in sectioned pellets and an inability to demonstrate the polygonal lattice with either the freeze-cleave or negative staining techniques. Extraction of the membranes with 50% acetone does not produce this effect. Thin-layer chromatography of the acetone extracts reveals a group of phospholipids in the 60% extract that are not detectable in the 50% extract. Acetone does not cause any detectable change in the structure of the zonula occludens, but the occluding junction becomes leaky to lanthanum following acetone treatment. The effects of other reagents on the junctions are reported.     

The use of lanthanum chloride as a marker for intercellular junctions in rat exocrine pancreas

A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly be electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues.     

Modulation of cell junctions during differentiation of the chicken otocyst sensory epithelium.

The differentiation of sensory and support cells within the embryonic chick otocyst is accompanied by alterations in the distribution of preexisting intercellular junctions. Prior to innervation of this epithelium, tight, gap and adhering junctions exist between all cells. Upon differentiation of the epithelium, apical bands of tight and adhering junctions are maintained throughout, while gap junctions and desmosomes are found only between support cells. Thus, some of the gap junctions that join homogeneous epithelial cells prior to innervation are removed as sensory cells differentiate, and a separate population of very large gap junctions is formed between differentiating support cells. Morphological evidence suggests two possible mechanisms which may be responsible for the observed changes in gap junctional distribution: removal of gap junctions by internalization, and formation of gap junctions by aggregation of precursor particles. The temporal correlation between junctional modulation, cytological differentiation of sensory and support cells, and ingrowth of nerve fibers makes the latter event a likely developmental cue for differentiation of this epithelium.     

COEXISTENCE OF GAP AND SEPTATE JUNCTIONS IN AN INVERTEBRATE EPITHELIUM

The intercellular junctions of the epithelium lining the hepatic caecum of Daphnia were examined. Electron microscope investigations involved both conventionally fixed material and tissue exposed to a lanthanum tracer of the extracellular space. Both septate junctions and gap junctions occur between the cells studied. The septate junctions lie apically and resemble those commonly discerned between cells of other invertebrates. They are atypical in that the high electron opacity of the extracellular space obscures septa in routine preparations. The gap junctions are characterized by a uniform 30 A space between apposed cell membranes. Lanthanum treatment of gap junctions reveals an array of particles of 95 A diameter and 120 A separation lying in the plane of the junction. As this pattern closely resembles that described previously in vertebrates, it appears that the gap junction is phylogenetically widespread. In view of evidence that the gap junction mediates intercellular electrotonic coupling, the assignment of a coupling role to other junctions, notably the septate junction, must be questioned wherever these junctions coexist.     

A DISTINCTIVE CELL CONTACT IN THE RAT ADRENAL CORTEX

Extensive cell contacts which resemble septate junctions occur between cells in the three major zones of the rat adrenal cortex. Characteristically, they extend between small intercellular canaliculi and the periendothelial space, frequently interrupted by gap junctions and rarely by desmosomes. Zonulae occludentes have not been identified in the adrenal cortex. Along this distinctive cell contact, the cell membranes of apposing cells are separated by 210–300 a bisected by irregularly spaced 100–150-A extracellular particles which are often circular in profile. In lanthanum preparations, these particles appear to form a continuous chain throughout the intercellular space and are visualized as an alveolate structure in sections parallel to the plane of the cell membrane. The cell membrane in the area of septate-like contact does not differ from nonjunctional areas of the cell membrane in freeze-fracture replicas. The cell contact retains its integrity after cell dispersion and after the separation of cell membranes from disrupted cells. The intercellular particles also persist after brief extraction in lipid solvents. Besides adherence, possible functions of this adrenal contact include maintenance of the width of the extracellular space, the provision of channels between intercellular canaliculi and the bloodstream, and utilization as cation depots. Similar structures are also present between adrenal cortical cells of several other species and between interstitial cells of the testis. This type of cell contact may, in fact, be a typical feature of steroid-hormone-secreting tissues in vertebrates.     

Electrophoretic repatterning of charged cytoplasmic molecules within tissues coupled by gap junctions by externally applied electric fields

Ionic currents and cytoplasmic voltage gradients have been observed in a variety of polarizing cells and developing tissues. In certain cases, it has been determined that these endogenous electric fields can segregate intracellular charged molecules by electrophoresis; in other cases, the endogenous fields are suspected to have such an influence. Separate theoretical suggestions have been made that extracellular electric currents, whether from a biological or a nonbiological source, should be able to electrophorese intracellular molecules after being conducted through cell membranes into the interior of long single cells [L.F. Jaffe and R. Nuccitelli (1977) Annu. Rev. Biophys. Bioeng. 6, 445-476] or extended ensembles of cells coupled electrotonically by gap junctions [M.S. Cooper (1984) J. Theor. Biol. 111, 123-130]. To test whether external electric fields could redistribute intracellular molecules within a tissue coupled by gap junctions, and to quantitatively measure in situ the electrophoretic mobility of a charged intracellular molecule, we injected 6-carboxyfluorescein into the electrotonically coupled lateral giant neurons of the crayfish abdominal nerve cord. When a dc electric field (0.2-3.4 V/cm) was subsequently applied along the length of the cord, the negatively charged fluorescent dye was observed to migrate through both the cytoplasms and the gap junctions of the lateral giant neurons, toward the anode, at a rate directly proportional to the applied electric field strength (electrophoretic mobility = -0.92 +/- 0.27 micron/sec per V/cm). These results suggest that electric fields of a sufficient magnitude, whether of an exogenous or an endogenous origin, can repattern the distribution of charged molecules within the cytoplasm of an extended ensemble of coupled cells. In addition, these results suggest that externally applied electric fields might be used in studies of pattern formation to repattern the intercellular distribution of charged molecules that are permeant to gap junctions within electrically coupled tissues.