Uptake of peroxidose by the cockroach central nervous system

The central nervous system of the cockroach has been incubated with solutions of an exogenous tracer substance, horseradish peroxidase, and the sites of its penetration and uptake have been studied by electron microscopy. When the nervous tissue is intact, or intact but stretched, the peroxidase is taken up throughout the neural lamella and also penetrates short distances into the extracellular space between adjacent perineurial cells. When the ganglia have been desheathed, reaction product for peroxidase is found in the neural lamella, perineurial cells, and within the cytoplasmic substance of the glial cells adjacent to the desheathed area. This uptake of peroxidase by the injured glial cells in desheathed preparations may reflect an alteration in the normal diffusion pathway from the external medium to the axonal surfaces.     

Ultrastructural pathology of the compound eye and optic neuropiles of the retinal degeneration mutant (w rdgB KS222) Drosophila melanogaster

Summary Lanthanum, applied to the outside of the fixed sciatic nerve of Rana pipiens, did not enter the endoneurium, but was halted by functionally tight junctions at the inner layers of the perineurium. This component of the bloodnerve barrier consists of several concentric layers of cells interspersed with an extracellular matrix of amorphous ground substance, collagen fibrils, and fine filaments. Numerous vesicular profiles are closely associated with the surface membranes of all the cells. The application of lanthanum to fixed tissue revealed that these profiles are attached to the cell surface by narrow necks, and open to the extracellular space. The attenuated cells are filled by the vesicular structures, which often appear to overlap. Stereoscopic electron microscopy showed that these vesicles did not fuse with each other or with the apposing cell surface to form transcellular channels. Channel formation does not appear to contribute significantly to the permeability of any of the perineurial layers.     

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     

Analog of vertebrate anionic sites in blood-brain interface of larval Drosophila

The blood-brain barrier ensures brain function in vertebrates and in some invertebrates by maintaining ionic integrity of the extraneuronal bathing fluid. Recent studies have demonstrated that anionic sites on the luminal surface of vascular endothelial cells collaborate with tight junctions to effect this barrier in vertebrates. We characterize these two analogous barrier factors for the first time on Drosophila larva by an electron-dense tracer and cationic gold labeling. Ionic lanthanum entered into but not through the extracellular channels between perineurial cells. Tracer is ultimately excluded from neurons in the ventral ganglion mainly by an extensive series of (pleated sheet) septate junctions between perineurial cells. Continuous junctions, a variant of the septate junction, were not as efficient as the pleated sheet variety in blocking tracer. An anionic domain now is demonstrated in Drosophila central nervous system through the use of cationic colloidal gold in LR White embedment. Anionic domains are specifically stationed in the neural lamella and not noted in the other cell levels of the blood-brain interface. It is proposed that in the central nervous system of the Drosophila larva the array of septate junctions between perineurial cells is the physical barrier, while the anionic domains in neural lamella are a charge-selective barrier for cations. All of these results are discussed relative to analogous characteristics of the vertebrate blood-brain barrier.     

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.     

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.     

Insect peripheral nerves: accessibility of neurohaemal regions to lanthanum.

During incubation in vivo, exogenously applied ionic lanthanum comes to surround the numerous neurosecretory terminals which are found lying within or immediately beneath the acellular neural lamella ensheathing the nerves from fifth instar and adult specimens of Rhodnius prolixus. The lanthanum does not penetrate beyond the cellular perineurium, which completely surrounds the non-neurosecretory axons in these nerves and constitutes a form of 'blood-brain barrier'. In some cases, however, lanthanum is found in the vicinity of a neurosecretory axon lying beneath the perineurium, where it can be assumed to have leaked in from the neurosecretory terminal lying free in the neural lamella. When nerves are incubated in calcium-free media, regions with an attenuated perineurium become 'leaky', in that lanthanum is found lying in those extracellular spaces between axons and glia which lie immediately below the thin part of the perineurial layer. Bathing solutions made slightly hyperosmotic to the haemolymph with sucrose have no apparent disruptive effects on the barrier. When the tissues are incubated in more hypertonic solutions, the perineurial barrier becomes 'leaky' throughout, and tracer pervades beyond its cells into all the intercellular spaced between glia and axons. The possible role of the zonulae occludentes in both the maintenance of the perineurial barrier and in the formation of interglial occlusions to local penetration of exogenous substances is considered.     

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.     

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.     

Pleated septate junctions in leech photoreceptors: ultrastructure, arrangement of septa, gate and fence functions

The leech photoreceptor forms a unicellular epithelium: every cell surrounds an extracellular “vacuole” that is connected to the remaining extracellular space via narrow clefts containing pleated septate junctions. We analyzed the complete structural layout of all septa within the junctional complex in elastic brightfield stereo electron micrographs of semithin serial sections from photoreceptors infiltrated with colloidal lanthanum. The septa form tortuous interseptal corridors that are spatially continuous, and open ended basally and apically. Individual septa seem to be impermeable to lanthanum; interseptal corridors form the only diffusional pathway for this ion. The junctions form no diffusion barrier for the electron-dense tracer Ba²⁺, but they hinder the diffusion of various hydrophilic fluorescent dyes as demonstrated by confocal laser scanning microscopy (CLSM) of live cells. Even those dyes that penetrate gap junctions do not diffuse beyond the septate junctions. The aqueous diffusion pathway within the septal corridors is, therefore, less permeable than the gap-junctional pore. Our morphological results combined with published electrophysiological data suggest that the septa themselves are not completely tight for small physiologically relevant ions. We also examined, by CLSM, whether the septate junctions create a permeability barrier for the lateral diffusion of fluorescent lipophilic dyes incorporated into the peripheral membrane domain. AFC₁₆, claimed to remain in the outer membrane leaflet, does not diffuse beyond the junctional region, whereas DiIC₁₆, claimed to flip-flop, does. Thus, pleated septate junctions, like vertebrate tight junctions, contribute to the maintenance of cell polarity.     

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.     

Establishment of a Convenient System for the Culture and Study of Perineurium Barrier In Vitro

The perineurium serves as a selective, metabolically active diffusion barrier in the peripheral nervous system, which is composed of perineurial cells joined together by tight junctions (TJs). Not only are these junctions known to play an essential role in maintaining cellular polarity and tissue integrity, but also limit the paracellular diffusion of certain molecules and ions, whereas loss of TJs barrier function is imperative for tumour growth, invasion and metastasis. Hence, a detailed study on the barrier function of perineurial cells may provide insights into the molecular mechanism of perineural invasion (PNI). In this study, we aimed to develop an efficient procedure for the establishment of perineurial cell lines as a tool for investigating the physiology and pathophysiology of the peripheral nerve barriers. Herein, the isolation, expansion, characterization and maintenance of perineurial cell lines under favourable conditions are presented. Furthermore, the analysis of the phenotypic features of these perineurial cells as well as the barrier function for the study of PNI are described. Such techniques may provide a valuable means for the functional and molecular investigation of perineurial cells, and in particular may elucidate the pathogenesis and progression of PNI, and other peripheral nerve disorders.

     

The passage of radioactive lanthanum from the biliary to the vascular system

Summary Radioactive lanthanum nitrate, an electron opaque tracer, was injected into the common bile duct of rats. Two minutes following the end of injection, samples of blood for radioactivity counts, and of liver and kidney for electron microscopic studies were taken. High levels of radioactivity found in the blood, and demonstration of lanthanum in the kidney by electron microscopy, indicated that this tracer entered the blood stream in vivo. No lanthanum was seen in the cytoplasm of liver cells, and there was no evidence of rupture in bile ducts, junctional ducts, or liver cells. Tight junctions connecting parenchymal cells and cholangiolar cells appeared well preserved. Lanthanum was seen in bile canaliculi, interspaces between liver cells, portions of the extracellular space of the liver, lumina of cholangioles and lumina of portal venules and sinusoids. It is postulated that lanthanum passed from the biliary tract, the site of injection, through the tight junction between liver cells and cholangiolar cells. It is suggested that such passage may represent a physiologic pathway, but the possibility of a chemical action of lanthanum on the tight junction can not be ruled out.This study was supported by the A. D. Williams Fund of Virginia Commonwealth University and the U. S. Veterans Administration. Radiation Counting Equipment was loaned by Dr. F. O'Foghludha, Department of Radiation Physics, Virginia Commonwealth University.     

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.     

The leech photoreceptor cell: ultrastructure of clefts connecting the phaosome with extracellular space demonstrated by lanthanum deposition

Summary When the extracellular space in eyes of Hirudo medicinalis was traced by means of lanthanum deposition, clefts were found which extend into receptor cells connecting the phaosome vacuoles with the extracellular space. The membrane of the phaosome, which bears the photoreceptor microvilli, is continuous with the external membrane of the receptor cell. Thus mechanisms by which the initial events of photoreception bring about electrical events at the cell surface need not differ from those being considered for other photoreceptor cells.Intracleft structures were revealed in negative constrast by the lanthanum deposits. Bridges join the opposed membranes on either side of a cleft. Isolated isodiametric profiles (120 A in diameter), and zig-zag linear structures, that are perhaps linear arrays of the isodiametric structures, were revealed in tangential sections of lanthanum filled clefts.Supported by the National Science Foundation, Grant GB-4822, and by the Deutsche Forschungsgemeinschaft.We thank Mrs. Carol Deuel Sundeen for technical assistance.     

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.     

Sustaining olfaction at low salinities: evidence for a paracellular route of ion movement from the hemolymph to the sensillar lymph in the olfactory sensilla of the blue crab Callinectes sapidus

Evidence reported previously suggests that in low-salinity conditions the integrity of the olfactory dendrites of the blue crab is sustained by a diffusion-generated ionic microenvironment within the aesthetascs. Diffusion of ions from the hemolymph to the sensillar lymph is proposed to maintain this microenvironment. In this study, using lanthanum as an electron-dense marker of extracellular fluid space, we find morphological evidence for paracellular continuity between the hemolymph and the sensillar lymph. Lanthanum penetrates extracellular fluid spaces within the aesthetascs when antennules are either perfused or bathed externally with solutions containing lanthanum nitrate. This was found in both freshwater- and seawater-acclimated animals. Evidence for ion diffusion from the aesthetascs was obtained using self-referencing, ion-selective microelectrodes. Both Ca2+ and K+ exhibit outwardly directed flux gradients associated with the aesthetasc tuft in low-salinity conditions. These findings are consistent with the concept that ion diffusion from the hemolymph to the sensillar lymph generates an ionic/osmotic microenvironment within the aesthetascs at low salinities.     

Incidence of Spinal Perineurial (Tarlov) Cysts among East-European Patients

The spinal perineurial cyst (Tarlov) is a dilatation between the perineurium and endoneurium of spinal nerve roots, located at level of the spinal ganglion and filled with cerebrospinal fluid but without communication with the perineurial subarachnoid space. The aim of the study was to evaluate it incidence among East-European patients. The retrospective data collected during various magnetic resonance spinal examinations and stored on the picture archiving and communication system was analyzed for an incidence of perineurial cysts. From among 842 patients that underwent examination, 75 cases perineurial cysts were revealed. In 22 cases single anomalies were found. In remaining 53 cases, multiple uni- or less frequently bilateral changes were noted. The most common position was the sacral canal, particularly the level of S2 and S3. Occasionally, cysts were also visible on the cervical, thoracic and lumbar level. Incidence of sacral perineurial cysts was significantly higher in females than in males. Similar data was found for single and multiple changes despite of their localization. Insignificant changes were seen for patient age and cyst size. Perineurial spinal cysts were the most frequently observed on the sacral level and such changes were more common in females.     

Effects of ATP on intracellular calcium dynamics of the perineurium of peripheral nerve bundles

Adenosine-5'-triphosphate (ATP) released from damaged cells can affect functions of adjacent cells. Injuries of peripheral tissue stimulate nerves, but effect of ATP on the nerve bundles is still speculative. Peripheral nerves are surrounded by perineurium, therefore the response of perineurium may be a first event of nerve stimulation at tissue injuries. The aim of the present study is to clarify whether the perineurium responds to ATP. To this end, we analyzed the dynamics of the intracellular calcium concentration ([Ca2+]i) of perineurial cells by confocal microscopy. ATP induced a [Ca2+]i increase of perineurial cells. Ca2+ channel blockers and removing of extracellular Ca2+, but not thapsigargin pretreatment, abolished ATP-induced [Ca2+]i dynamics. This indicated that the [Ca2+]i increase was due to an influx of extracellular Ca2+. Adenosine-5'-diphosphate also elicited an increase of [Ca2+]i, but P1 receptor agonists had few effects on [Ca2+]i dynamics. Suramin (an antagonist of P2X and P2Y receptors) totally inhibited ATP-induced [Ca2+]i dynamics, but reactive blue 2 (a P2Y receptor antagonist) did not. Uridine-5'-triphosphate (a P2Y receptor agonist) induced no significant change in [Ca2+]i, but alpha,beta-methylene ATP (a P2X receptor agonist) caused a [Ca2+]i increase. In conclusion, perineurial cells respond to extracellular ATP mainly via P2X receptors.