Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions

In the central nervous system (CNS) of pupal Calliphora, dramatic alterations occur in the perineurial and glial gap junctions. Having formed macular plaques by late larval stages, in early pupae cell migration causes the EF intramembranous junctional particles to disaggregate and move apart into linear and then disorganised arrays as shown by freeze-fracture. After nerve and glial cell reorganisation into the adult pattern, the gap junctions begin to reform in the late pupae, again seemingly by particle migration into linear arrays and clusters. Ultimately the particles form numerous macular plaques between both perineurial and glial cells. Statistical analyses support the contention that these are performed EF particles which undergo translateral movement from macular larval junctions into the disaggregated particles of early pupae and that the same particles appear to undergo realignment and reclustering in late pupae to form the mature gap junctions of adults. This is the first report to indicate breakdown and reformation of gap junctions in vivo involving reutilisation of the same intramembranous particles. Perineurial “tight” junctions are not to be found in early pupal stages and their absence can be correlated with the free entry of ionic lanthanum into the CNS observed during that period. In late pupae, when the tight junctional moniliform ridges have apparently reformed, the entry of the tracer lanthanum becomes restricted to the level of the perineurium, penetrating no deeper. This is also the case in the adult, where the blood-brain barrier is maintained. PF particles in the form of short linear ridges and clustered particle arrays in nerve cell membranes are present throughout pupal and adult stages; their continued presence throughout the whole of development suggests some role in neuronal function, as yet unclear.     

Glial cells in peripheral nerves of the cockroach, Periplaneta americana

The ultrastructural organization and the junctional complexes of peripheral nerves have been investigated in the cockroach Periplaneta americana. Nerve 5 is surrounded by a layer of connective tissue, the neural lamella, beneath which is a layer of perineurial glial cells wrapping the axons. Adjacent perineurial cells are joined to one another by septate, gap and tight junctions. Septate and gap junctions were observed in freeze-fracture replicas of main trunk nerve 5. Septate junctions were found as rows of PF particles mainly in perineurial cell membranes. Gap junctions exhibited EF macular aggregates in perineurial and subperineurial glial cells. During incubations in vivo with extracellularly applied ionic lanthanum, the lanthanum did not penetrate beyond the perineurium. Where nerve 5 branches and contacts the muscle, lanthanum penetrated freely between the muscle fibres and the nerve branches. In small peripheral branches where the axons are surrounded by single a glial layer, lanthanum is unable to penetrate to the axolemma.     

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.     

A novel adhering junction in the apical ciliary apparatus of the rotifer Brachionus plicatilis (Rotifera, Monogononta)

Cultures of the rotifer Brachionus plicatilis were examined with regard to their interepithelial junctions after infiltration with the extracellular tracer lanthanum, freeze-fracturing or quick-freeze deepetching. The lateral borders between ciliated cells have an unusual apical adhering junction. This apical part of their intercellular cleft looks desmosome-like, but it is characterized by unusual intramembranous E-face clusters of particles. Deep-etching reveals that these are packed together in short rows which lie parallel to one another in orderly arrays. The true membrane surface in these areas features filaments in the form of short ribbons; these are produced by projections, possibly part of the glycocalyx, emerging from the membranes, between which the electron-dense tracer lanthanum permeates. These projections appear to overlap with each other in the centre of the intercellular cleft; this would provide a particularly flexible adaptation to maintain cell-cell contact and coordination as a consequence. The filamentous ribbons may be held in position by the intramembranous particle arrays since both have a similar size and distribution. These contacts are quite different from desmosomes and appear to represent a distinct new category of adhesive cell-cell junction. Beneath these novel structures, conventional pleated septate junctions are found, exhibiting the undulating intercellular ribbons typical of this junctional type, as well as the usual parallel alignments of intramembranous rows of EF grooves and PF particles. Below these are found gap junctions as close-packed plaques of intramembranous particles on either the P-face or E-face. After freeze-fracturing, the complementary fracture face to the particles shows pits, usually on the P-face, arrayed with a very precise hexagonal pattern.     

Vertebrate-like tight junctions in the insect eye

Unequivocal vertebrate-like anastomosing tight junctions have been observed for the first time in insect tissues. In freeze-fractured replicas of dipteran compound eyes, the intercellular junctions between certain glial cells in regions distal to the optic neuropile display an extensive network of continuous intramembranous P face (PF) ridges. The intramembranous E face (EF) possesses a reticulum of grooves which occur in the depths of troughs and thereby produce a ‘quilted’ appearance. At PF/EF membrane face transitions, there is an obliteration of the intercellular space at points of membrane fusion; here the PF ridges and EF grooves appear in register and are therefore complementary. Although the septate junctions found here are patent, these tight junctions are occluding to lanthanum and appear to represent the blood-retinal barrier previously demonstrated electrophysiologically in insects. The existence and vertebrate-like structural complexity of these junctions in arthropods supports the concept of the universality of the membrane specializations that mediate cell-to-cell interactions.     

The penetration of ionic lanthanum into the central nervous system of the tick, Amblyomma variegatum

ABSTRACT. The ultrastructure of the tick central nervous system resembles that of insects except that the perineurial layer of specialized glial cells is less well developed in the tick. In particular, the cells are not connected by tight or septate junctions. Probably as a consequence, ionic lanthanum penetrates the entire central nervous system of the tick, whereas it fails to penetrate the perineurium of insects. These observations suggest that ticks lack the 'blood—brain barrier' which protects the insect nervous system.     

The perineurium of the adult housefly: Ultrastructure and permeability to lanthanum

Summary The ultrastructure of the perineurial cells of Musca overlying the first optic neuropile was examined by transmission electron microscopy. These cells are somewhat similar to those of other insects but cytoplasmic flanges seem to be absent, and mitochondria are relatively large and sinuous. The intercellular channel system on the lateral border of the cells is relatively spacious and highly meandering. Perineurial cells are joined by septate, gap, and tight junctions, hemidesmosomes, and desmosomes. Tight and septate junctions bond perineurial cells and glial cells. These data are evaluated on the basis of tracer studies with lanthanum. This material penetrates the extracellular space between perineurium and underlying glial and nerve cells, between epithelial glial cells and retinular axon terminals (capitate projections), and between the - fiber pair in the optic cartridge (gnarls). If no damage occurs to the perineurial cells during tissue preparation, this passage of lanthanum to neuronal surfaces indicates that the blood brain barrier is incomplete in this restricted area. Supportive evidence for such permeance is based on electrophysiological data, considerations of membrane specializations in the optic neuropile, and Na⁺/K⁺ ratios of dipteran hemolymph.We gratefully acknowledge support from the N.I.H., National Eye Institute, EYO 1686 and from the College of Agricultural and Life Sciences, Hatch Project 2100. Richard L. St. Marie and Professor Stanley D. Beck, Department of Entomology, UW, Madison read early drafts of this paper and provided constructive comments     

X-ray microanalysis with transmission electron microscopy determined presence and movement of tracer (lanthanum chloride) at blood-neuron barrier of Drosophila melanogaster (Diptera : Drosophilidae) larva

In studying the larval Drosophila (Diptera : Drosophilidae) blood-brain barrier, it was important to determine if even minute amounts of tracer ultimately seeped through the septate junctions between perineurial cells to reach the neuronal region. Concurrent TEM with X-ray microanalysis was undertaken to resolve that issue. Ultrathin sections of Drosophila nervous tissue in LR White embedment were exposed to ionic tracer (lanthanum chloride) and assayed for presence or absence of lanthanum extracellular to the perineurium and glia making up the nerve sheath. Tracer filled the distal interseptal lattice of pleated sheet-septate junctions, but was contained prior to reaching the proximal paracellular space. No detectable tracer passed through septate junctions to enter the glial-neuronal domain. Based on our present data and the research of others, septate junctions in immature Drosophila are multifunctional structures that enforce spatial relationships between cells, seal intercellular spaces, and control cell proliferation in the epithelia. Septate junctions in Drosophila with the (dlg) gene also exhibit protein homologies to the Z0–1 human tight junction component.     

Intercellular junctions and rhombic particle arrays in the developing and adult dorsal ocelli of the honeybee

Intercellular junctions and particle arrays in the developing and mature dorsal ocelli of the honeybee Apis mellifera have been studied with conventional and freeze-fracture electron microscopy. Four types of junctions are found in the lentigenic and retinogenic part during development. These are desmosomes, septate junctions, tight junctions, and gap junctions. Gap junctions and septate junctions are found between differentiating photoreceptor cells only as long as the rhabdoms are beginning to form. Their disappearance after differentiation indicates that they could play a part in cell determination. Desmosomes connect photoreceptor cells into the early imaginai stage and then disappear. Other junctions, once they have formed, remain for the life of the animal, but can change considerably in structure, distribution and frequency. The cells of the perineurium surrounding the ocellus are connected by septate and gap junctions, which may be the basis of the blood-eye barrier. Rhombic particle arrays on the E-face of the glial membrane attached to the photoreceptor cell membrane first appear in small groups one day before emergence. In the further course of life these arrays become more extensive and apparent. Their significance may be to play some role in receptor function.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

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.     

Cell junctions in the gut of Protura

The main cell junctions in the intestinal tract of a small group of apterygotan insects, Protura, were examined in conventional thin sections, tracer-infiltrated sections and freeze-fracture replicas. The smooth septate junctions in the midgut of collembolan Tomocerus minor were also studied for comparison. Pleated septate junctions are found in foregut, hindgut and Malpighian papillae. They exhibit regular septa crossing the intercellular clefts in thin sections; and the septa with a pronounced zig-zag appearance run parallel to form a honeycomb structure in tracer-impregnated sections. After freeze-fracture undulating rows of intramembranous particles (IMPs) are visible on the P face. Smooth septate juncions are observed in the midgut. The interceullar septa often run in pairs for long tracts and exhibit a wavy course in lanthanum impregnated sections. The IMPs exhibited on the E face are usually separated one from another. Twin arrangement of particle rows is also apparent on the replicas. Gap junctions are frequent in both the midgut and hindgut and possess the conventional characteristics of 'inverted gap junction' with E face connexons. These results provide further evidence relating Protura closely to Collembola as well as to primitive arthropods.     

Glial cell contacts in insects: Effects of feeding on intercellular junctions

The intercellular junctions associated with the modified glial cells of the perineurium have been examined in the ganglia and main abdominal nerves of the blood-sucking bug Rhodnius prolixus, both before and and after feeding, by means of freeze-fracture and tracer studies. It was found that the pleated septate junctions found in the main abdominal nerve have many fewer septa than those found in the ganglion. These junctions appear to provide the flexibility needed for the movement of cells which occurs to accommodate the tremendous increase in body size that takes place after a bloodmeal. On feeding and during the subsequent period of digestion the nerves stretch to double their length, yet the blood-brain barrier is maintained throughout. In the same manner as loosely interconnected tight junctions, septate junctions with fewer septa seem to form a junction which is able to respond readily to the stress of stretching. With feeding and afterwards the septate junctions become disorganized and disassemble, while the gap junctions and tight junctions remain intact. It is envisaged, therefore, that the primary function of the septate junction is adhesive.     

Rhombic particle arrays in gill epithelium of a mollusc, Aplysia californica.

Gill epithelia from adult and juvenile Aplysia were examined by conventional thin section and freeze-fracture methods. Freeze-fracture replicas of adult gill epithelium revealed septate and gap junctions, which served as membrane markers for the epithelial cells. In these same cell membranes, non-junctional rhombic arrays of intramembranous particles were observed on prominent ridges on the membrane P fracture face of some epithelial cells. In thin sections of adult epithelium, nerve terminals were observed abutting the lateral plasma membranes near the basal lamina of some epithelial cells. Correlative areas of plasma membrane in freeze-fracture replicas showed a close association between rhombic particle arrays and abutting nerve terminals. In thin sections of juvenile Aplysia, nerve terminals abutting the epithelial cells were not recognizable, and rhombic arrays were not observed in freeze-fracture replicas. This suggested that a developmental association existed between the appearance of rhombic arrays in adult epithelia and their innervation. It is not known with certainty if, in invertebrates, rhombic arrays are an essential structural entity of all innervated cell membranes; however, in the cells thus far studied, there appears to be an associative condition. In the case of the gill epithelium of Aplysia, rhombic arrays are located in the same vicinity as the abutting nerve terminals. Similar arrays of intramembranous particles have been observed in myoneural postjunctional complexes of other invertebrates and have been interpreted to be the morphological expression of neurotransmitter receptors. An analogous explanation is put forth, namely that rhombic arrays may represent the structural correlates of neurotransmitter receptors and/or ionic channels in innervated membranes of invertebrates.     

Fine structures of sponge cell membranes: comparative study with freeze-fracture and conventional thin section methods

Freeze-fracture replicas of sponge cell membranes revealed in general a low density of intramembranous particles, with the exceptions of the membrane (silicalemma) surrounding the siliceous spicules in Ephydatia and the membranes of spherulous cells in Chondrosia. In addition, several types of particle arrangements were observed. A classical necklace is present at the base of the choanocyte flagellum. Rosettes of particles are particularly obvious in the apical membranes of choanocytes, where they are associated with the fuzzy coat covering these cells. Parallel ridges of particles were observed along the microvilli of the choanocyte collar, at sites of insertion of connecting filaments. Rows of particles were observed in the plasma membrane of pinacocytes in Ephydatia where they are located on areas deformed by protruding fibrillar inclusions. Pinacocyte plasma membranes in this species also can contain accumulations of particles which are likely related to desmosomes. Single rows of aligned particles and double rows of staggered particles (sometimes organized in large plates) in addition to rhombic particle arrays were encountered on replicas of marine sponge cell membranes. No classical arrangements corresponding to gap junctions, tight junctions or septate desmosomes were observed. The significance of these data is analysed.     

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.     

A blood-brain barrier without tight junctions in the fly central nervous system in the early postembryonic stage

Summary The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.     

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.     

Perineurium in the Drosophila (Diptera : Drosophilidae) embryo and its role in the blood-brain/nerve barrier

A monolayer of perineurial cells overlies glia and neurons, and this stratum of the central nervous system is the principal site of the Drosophila (Diptera : Drosophilidae) blood-brain barrier. Perineurial cells are bonded together by pleated-sheet septate junctions that are the anatomical correlate of the vertebrate tight junction. The blood-brain barrier maintains the ionic homeostasis necessary for proper nerve function. It was known that a functioning blood-brain barrier is present in mature (Stage 17) Drosophila embryos, but the genesis of this barrier was not known. We surveyed the central nervous system of late stage embryos (15 through 17) to determine when perineurial cells could first be detected. These cells take their place in (on) the central nervous system and are joined together by pleated-sheet septate junctions, during Stage 17. Those septate junctions are quickly occlusive to lanthanum tracer. This development step occurs during the same time as when chemical synapses first become functional. Such concurrent maturation is far from coincidental, because partitioning nerves and their synapses from hemolymph (with its variable ionic constitution) are essential for normal electrophysiology. We discuss details of the germ line derivation of perineurial cells, their first detection in the embryonic central nervous system, their functional properties, and the polygonal cell-packing pattern seen in the larval central nervous system.     

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca,Pulmonata)

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.