insect peripheral nerves: accessibility of neurohaemal...

J. Cell Sci. 18, I79-I97 (i975) 179Printed in Great Britain

INSECT PERIPHERAL NERVES: ACCESSIBILITY

OF NEUROHAEMAL REGIONS TO

LANTHANUM

NANCY J. LANE, R. A. LESLIE AND LESLEY S. SWALES

Agricultural Research Council Unit of Invertebrate Chemistry andPhysiology, Department of Zoology, Univertity of Cambridge,Dotoning Street, Cambridge CBz 3EJ, England

SUMMARY

During incubation in vivo, exogenously applied ionic lanthanum comes to surround thenumerous neurosecretory terminals which are found lying within or immediately beneaththe acellular neural lamella ensheathing the nerves from fifth instar and adult specimens ofRhodnius prolixus. The lanthanum does not penetrate beyond the cellular perineurium, whichcompletely surrounds the non-neurosecretory axons in these nerves and constitutes a formof 'blood-brain barrier'. In some cases, however, lanthanum is found in the vicinity of aneurosecretory axon lying beneath the perineurium, where it can be assumed to have leakedin from the neurosecretory terminal lying free in the neural lamella.

When nerves are incubated in calcium-free media, regions with an attenuated perineuriumbecome 'leaky', in that lanthanum is found lying in those extracellular spaces between axonsand glia which lie immediately below the thin part of the perineurial layer. Bathing solutionsmade slightly hyperosmotic to the haemolymph with sucrose have no apparent disruptiveeffects on the barrier. When the tissues are incubated in more hypertonic solutions, theperineurial barrier becomes 'leaky' throughout, and tracer pervades beyond its cells into allthe intercellular spaces between glia and axons.

The possible role of the zonulae occludentes in both the maintenance of the perineurialbarrier and in the formation of interglial occlusions to local penetration of exogenous sub-stances is considered.

INTRODUCTION

It is well known that insect nervous systems have no blood vascular supply, sothat substances can gain access to the nerve cells only by diffusing through thesheaths that surround them. Recently it has become apparent that the sheaths maypresent restriction to entry of certain substances, suggesting the existence of aninsect 'blood-brain barrier' (Hoyle, 1952, 1953; Twarog & Roeder, 1956; Treherne& Pichon, 1972; Treherne, 1974). This barrier, preventing the free interchange ofions between the central nervous system and the blood, occurs both in phytophagousinsects, in which the haemolymph contains such low concentrations of sodium thatit would not permit the propagation of action potentials in the conventionalfashion (Treherne, 1965 a, b), and in insects such as the omnivorous cockroach,whose blood composition is ionically favourable as an immediate bathing mediumfor the nervous system (for example, Treherne, 1961, 1962; Lane & Treherne,1972).

180 N. J. Lane, R. A. Leslie and L. S. Swales

The structural basis of the insect blood-brain barrier is thought to be the zonulaeoccludentes ('tight junctions') between the cells of the perineurium. The perineurialsheath is a specialized cell layer which surrounds the glial-ensheathed axonal massof both the central nervous system and larger peripheral nerves with a continuouscellular envelope (Wigglesworth, 1959a, b\ Lane, 1972, 1974; Lane & Treherne,1973). The morphology of this sheath is very complex, since there are tortuous andextensive interdigitations between the adjacent component cells, the lateral bordersof which are linked by gap junctions, septate junctions (desmosomes) and zonulaeoccludentes. It is difficult to determine the extent, precise arrangement and inter-relationships of these junctions because of the complicated architecture of the cells.Recent studies have indicated that exogenous tracers can penetrate the septate andgap junctions of this sheath (Lane & Treherne, 1972; Treherne, Schofield & Lane,1973). Since, however, they do not permeate beyond the perineurial layer it hasbeen assumed that it is the zonulae occludentes between these cells which representthe structural basis of the barrier (Lane & Treherne, 1972).

Attempts have been made both in vertebrates (Rapoport, Hori & Klatzo, 1972)and in invertebrates (Treherne et al. 1973) to disrupt the blood-brain barrierexperimentally. Rapoport and his co-workers used high (2 M and 3 M) concentrationsof compounds of low lipid solubility topically applied to the neurovascular tissue ofrabbits (Rapoport et al. 1972) or perfused directly into the blood vascular systemof Rhesus monkeys (Rapoport & Thompson, 1973) to open the vascular endotheliumto Evans blue. Treherne et al. (1973) tried a similar set of experiments with thecockroach ventral nerve cord using short pulses of hyperosmotic urea or glucose inthe bathing solution before testing for disruption of the barrier. Using electro-physiological methods, they found that urea was effective in accelerating influx ofpotassium and efflux of sodium from these central nervous tissues, but were unableto find any differences in leakage of exogenously applied ionic lanthanum acrossthe blood-brain barrier following treatment with hypertonic urea or glucosesolutions.

Not only the central nervous systems of those insects studied, however, exhibita blood-brain barrier. Peripheral nerves, which are also bathed by the blood, containaxons which exhibit the same mode of nervous conduction as those of the centralnervous system, and large nerves of the locust and cockroach display a restrictionto entry of ions and molecules (Pichon & Treherne, 1973). In some very smallperipheral nerves of the cockroach, however, no ultrastructural basis for a barrierhas been found (Lane & Treherne, 1973).

It would seem worth while, therefore, to consider peripheral nerves in other systemsand the blood-sucking bug Rhodnius prolixus was chosen because it has a fusedthoracic ganglionic mass from which emanate many peripheral nerves. Moreover, ithas been shown (Maddrell, 1966) that these abdominal nerves in 5th instar larvaof this insect have many neurosecretory terminals which could represent a situationparallel to that of the small peripheral nerves of the cockroach. This report presentsevidence to show that, although the normal peripheral nerves in Rhodnius displaya blood-brain barrier by presenting a restriction to entry of exogenous tracers, the

Neurohaemal regions in insect nerves 181

neurosecretory endings are freely accessible to ionic lanthanum when this electron-opaque substance is introduced into the bathing medium of the nervous system.Moreover, the blood-brain barrier existing in the central and peripheral nervoussystems of Rhodnius can be disrupted by certain hypertonic solutions or by modi-fication of the ionic environment. A preliminary report of these observations hasbeen published elsewhere (Lane, Leslie & Swales, 1974).

MATERIALS AND METHODS

Both adult and 5th instar specimens of Rhodnius prolixus Stal were used in this investigation.The tissues examined included the mesothoracic ganglionic mass and the associated abdominalnerves. For conventional preparation and examination of fine-structural features the gangliaand nerves were fixed in 3 % glutaraldehyde in 0-05 M phosphate buffer, pH 6-9, plus i '7%sucrose. This was followed by several washes in 0-05 M cacodylate buffer+12% sucrose,then treatment with 1% osmium tetroxide in 0-05 M cacodylate buffer+io-3% sucrose,a brief wash in buffer plus 12% sucrose, and staining in 2 % uranyl acetate in sodium hydrogenmaleate buffer, pH 62, for 30 min. In certain control preparations this uranyl acetate stainingwas omitted. In some cases, sections were stained with phosphotungstic acid (PTA).

Other tissues were incubated in phosphate-free physiological saline to which lanthanumat different concentrations had been added. This saline, containing NaCl, 129 mM; KC1,8-6 mM; MgCls, 8-5 mM; CaCL, 2-0 mM; glucose, 34 mM; and Tris-HCl, 15 mM, at pH 7-5,is approximately 365 milliosmolal (modified from Maddrell, 1969; Farmer, personalcommunication). Lanthanum chloride at concentrations of 1-5 mM was routinely added tothis, although in some experiments higher concentrations were added, and the resultingsolutions were used to incubate the tissues for 30, 60 or 120 min. Controls were incubatedin saline to which no lanthanum was added. After incubation the tissues were fixed inphosphate-buffered glutaraldehyde to accomplish precipitation of the lanthanum and thenprocessed as described above.

Other experimental tissues were treated in one of a variety of ways in an attempt to disruptthe blood-brain barrier, and were then incubated in solutions containing different concen-trations of lanthanum for varying periods of time. This was done in order to determinewhether the barrier had in fact been opened, as would be indicated by penetration of lanthanumbeyond the zonulae occhtdentes of the perineurial barrier (Neaves, 1973). The treatmentsincluded incubating tissue in (1) 0-5 M sucrose for 2 min, or (2) 5 mM EGTA for 5 min.These were followed by a wash in calcium-free saline, and further incubation in low con-centrations of lanthanum chloride in phosphate-free (and in the case of EGTA treatment,calcium-free as well) saline, for 30-120 min. Tissue was also incubated in (3) calcium-freeand phosphate-free saline to which lanthanum chloride had been added. Control tissues weretreated with sucrose or EGTA followed by fixation, or subsequent incubation in phosphate-free or calcium-free physiological saline without added lanthanum, followed by fixation andembedding as described above.

RESULTS

Normal unincubated nervous tissue

In both adult and 5th instar Rhodnius, as many as 12-14 nerves of varying diameterare associated with the thoracic ganglionic mass; they are ensheathed by an acellularneural lamella under which lies a perineurial layer. The neural lamella consists ofcollagen-like fibrils embedded in an amorphous matrix; the fibrils exhibit a majorperiodicity of about 40-50 nm, but this banding is not very clear and is not enhancedby PTA staining as would be expected for collagen-like fibrils. The indistinct qualityof the banding pattern is similar to that found in the Lepidoptera and contrasts with


the clear periodic bands observed in cockroach, locust and stick insect (see Lane,

The perineurium is composed of flattened cells which may interdigitate with oneanother in a complex fashion. In some cases, however, the layer is extremelyattenuated (Figs. 6, 14). The cells may exhibit hemidesmosomes at their neurallamellar surface (Fig. 1). The lateral borders of adjacent cells are associated byjunctional complexes; these include septate junctions (septate desmosomes), gapjunctions and zonulae occludentes (Figs, i, 5, 7, 9, 11). In the ganglion the perineurialcells are much less flattened than in the nerves, and their nuclei appear circularrather than elliptical in outline; these cells are very much more voluminous thanthose of the nerves where they may be exceedingly attenuated. However, certainunusual features of the perineurium and the underlying glial and nervous tissues inthe ganglion will be reported more extensively elsewhere (N. J. Lane & L. S. Swales,in preparation).

The septate junctions between perineurial cells in the nerves seem to be relativelydisorganized, in that they often consist of short lengths with septa, alternating withnormal intercellular spaces where the plasma membranes are undifferentiated (Figs.5, 11). In those cases where the septa are not evident but the membranes are separatedby a regular space (Fig. 5) they may actually be 'continuous junctions' (Noirot &Noirot-Timothie, 1967; Satir & Gilula, 1973; Skaer & Lane, 1974). Septate junctionsare often found lying beneath areas where elliptical perineurial nuclei or neuro-secretory endings occur (Fig. 1).

Gap junctions are found at irregular intervals (Fig. 9) and they can be seen toextend over quite considerable lengths. They are characterized by the usual 2-3 nmgap (see Revel & Karnovsky, 1967) between adjacent perineurial plasma membranesbut the extent of each macular array has not yet been determined.

The zonulae occludentes in the perineurium appear to be mainly punctate (Fig. 7 A,B) in that extensive lengths of pentalaminar structures are rarely observed. Theextent of their complexity with respect to their surface characterization must awaitexamination by freeze-cleave techniques.

Beneath the perineurium of the peripheral nerves lie glial cells closely ensheathingthe axons. It appears that the larger the nerve in which they occur, the morevoluminous and laden with microtubules are the component glial cells. When verynumerous, the microtubules are packed closely together in a regular array in thecytoplasm (Fig. 17), all usually being oriented parallel to the longitudinal axis ofthe nerve. Glial cells vary in density; in some cases they are so opaque that themicrotubules stand out as clear areas. The glial cells also contain many mitochondria,and in regions where their cytoplasmic volume is substantial, they contain roughendoplasmic reticulum and Golgi complexes. Dense gliosomes (lysosomes) are alsooccasionally found. In some cases the glial cells appear to be associated with oneanother by gap junctions (Fig. 8) or zonulae occludentes (inset in Fig. 17). They maydisplay hemidesmosomes where they abut on to an extracellular space beneath theperineurium (Fig. 6).

The axons within the glial envestment usually contain mitochondria, neurotubules,


and smooth vesicles. However, they may also possess dense granules (Fig. 4) withthe characteristics of neurosecretory granules. These display a very considerablerange in diameter and have a core which is variable in opacity; they can sometimesbe seen to be separated from their delimiting membrane by a space or 'halo'.

In the 5th instar larvae, these abdominal nerves possess numerous neurohaemalareas (Fig. 1) which lie near the surface of the nerves (Maddrell, 1966). These areencountered very much less frequently in adults. They consist of axon terminals inthe neural lamella containing many neurosecretory granules with perineurial cyto-plasm beneath their substance but not covering them (Figs. 1-4, 13, 15).

Nervous tissue incubated in lanthanum solutions

Ganglia and nerves. In the ganglia of both 5th instar and adult insects, lanthanumdoes not penetrate beyond the basal border of the perineurial clefts, but readilypermeates the neural lamella and the extracellular spaces between adjacent perineurialcells. Similarly the peripheral nerves of the adults, as well as 5th instar specimens,display a restriction to the entry of ionic lanthanum, the tracer being found nodeeper than the intercellular perineurial clefts (Figs. 2, 3, 13, 15, 16). Both septatejunctions (Fig. 12) and gap junctions (Fig. 10) in the perineurium can be foundfull of lanthanum. Because of the frequently tortuous nature of these channels(Fig. 5) it is extremely difficult to determine exactly where the lanthanum stops, butsome preparations suggest that it is the zonulae occludentes which are responsiblefor restricting its further passage (Figs. 8, 12). Cells containing tracheoles, lyingin the perineurium, also become surrounded by lanthanum after incubation in thetracer (Fig. 15).

Control preparations incubated in saline without added lanthanum show no densedeposits in the perineurial clefts (Fig. 5). It is also apparent that fat body cells lyingbeyond the neural lamella play no occluding role since the lanthanum readilypenetrates beyond them to the underlying neural lamella and perineurium (Figs. 13,

H)-Neurohaemal regions. In the neurohaemal regions of the 5th instar animals, where

swollen neurosecretory endings are found lying near the nerve surface apparentlyreleasing their contents into the haemolymph (Maddrell, 1966, 1967, 1970, 1974),ionic lanthanum has free access to the endings, often penetrating completely aroundthem (Figs. 2, 3, 4, 13, 15). Sometimes a neurosecretory-laden axon, having notyet attained its point of release, can be found lying beneath the perineurium withlanthanum, surprisingly, also around it (Fig. 4). Presumably here the lanthanumhas leaked in from the neurosecretory terminal via the restricted extracellular spacearound the neurosecretory axon, since it seems unlikely to have progressed to thatpoint via normal perineurial channels, which ordinarily prevent the further entryof tracer (Figs. 2, 3). However, the lanthanum is only found in close proximity tothe neurosecretory axon, so it appears to be a very local diffusion of the tracer. Itis possible, therefore, that it is the zonulae occludentes between glial cells that arestopping the lanthanum from penetrating any further into the nervous system andsome micrographs suggest that this may be the case (Fig. 4).

12-3


Experimentally disrupted preparations

Calcium-free physiological saline treatments. In tissues incubated in calcium-freesaline to which lanthanum was added it can be seen that, although in many regionsthe perineurium does not permit entry of ionic lanthanum (Fig. 13), in some casesthe lanthanum does progress beyond the perineurium, past its junctional complexesinto the extracellular spaces between the glial cells ensheathing the axons (Fig. 14).This is a relatively infrequent occurrence and appears to be, in all cases, associatedwith a region where the perineurium is exceptionally thin. Possibly, therefore, theremay be less surface area of overlap between adjacent perineurial cells and hencefewer zonulae occludentes. Within the lanthanum-invaded nervous system, there aresuggestions (inset in Fig. 14) that the tracer is prevented from moving throughoutthe system by zonulae occludentes between adjacent glial cells.

Treatment with other solutions. When preparations are treated with sucrose forshort periods of time prior to incubation in lanthanum, the blood-brain barrier isnot disrupted (Fig. 15). It would appear, therefore, that the perineurial junctions areunaffected by these concentrations of sucrose and are still able to restrict the entryof the exogenous tracer. In EGTA-treated tissues, no invasion of the subperineurialspace has been found. Possibly the relative infrequency of any invasion of tracermake encountering it a rare event, as is the case with other tissues incubated incalcium-free saline (see preceding paragraph) (Fig. 14).

Hypertonic concentrations of lanthanum. When Rhodnius nerves are incubated inconcentrations of lanthanum as high as iomM (Fig. 15) or lOOmM (Fig. 16), thetracer is still unable to penetrate beyond the perineurium. Only when extremelyhigh concentrations of lanthanum are applied to the nervous system can the blood-brain barrier be disrupted (Figs. 18, 19). This occurs, though, only after the tissueshave been incubated in a presumably physiologically damaging 500 mil solutionfor longer than 30 min, when the lanthanum penetrates beyond the perineuriumand invades the intercellular spaces between glial cells in a dramatic fashion (Figs.18, 19). Under these circumstances certain microtubules found in the glial cellsbecome filled with lanthanum as well (Fig. 19). This may imply that under theseextreme and non-physiological conditions a physical association, possibly transitory,between the core of such microtubules and the lanthanum-filled extracellular spaceshas occurred, perhaps due to membrane damage induced by the high concentrationsof lanthanum.

DISCUSSION

It is clear from these experimental results that there is a restriction to entry of exo-genous tracers in the peripheral nerves of Rhodnius. The nerves examined, however,are all structurally comparable to the larger peripheral nerves of the cockroach (Lane &Treherne, 1973), which also display a 'barrier' in that they have been found topossess an extensive perineurium which contains occluding junctions. It is apparentthat ionic lanthanum easily penetrates the septate and gap junctions in the perineurium


and in this respect the situation is comparable to that of some vertebrates, where gapjunctions have also been found to permit entry of lanthanum (Revel & Karnovsky,1967). The evidence suggests that it is the zonulae occludentes which prevent themovement of the lanthanum beyond the level of the insect perineurium. It is difficultin view of the complexity of the perineurial fine structure to ascertain unequivocallyif this is true, although there are some micrographs (such as Fig. 8) which suggestthat it may be. Moreover, without detailed en face views by freeze-cleaving, theextent of the tight junctions (that is whether they are truly zonular or merely fasciaror macular) cannot be determined. Preliminary freeze-cleave studies on the peri-neurium of the locust indicate that the tight junctions in this insect may not exhibitas extensive an interconnecting meshwork as do the ridges or fibrils comprising thezonulae occludentes of vertebrate tissue (McNutt & Weinstein, 1973; Skaer & Lane,1974). However, in spite of this, insect perineurial junctions are obviously functionallyrelatively 'tight', rather than 'leaky' as appears to be the case with some vertebrateepithelia which also exhibit less numerous junctional fibrils (Claude & Goodenough,1973). In insects, the fact that the junctions are tight may be due to the extremecomplexity of the interdigitating perineurial processes so that even a simple system ofridges when interwoven in this way would be effective in occluding the inwardpassage of molecules.

Lanthanum is well established as a useful extracellular tracer molecule and isparticularly convenient because it is much smaller than many other available tracers,such as, for example, horseradish peroxidase (mol. wt. 40000). However, it should beborne in mind that lanthanum is a trivalent cation with a hydrated diameter of 2-78 nm(as measured at a concentration of 2 mM by Stern & Amis, 1959). This charge couldmean that it may bind at sites within the perineurium. Moreover, its size also meansthat the distribution and pattern of lanthanum uptake may not in fact reveal thesubtle changes that could occur in intercellular permeability involving physiologicallyimportant, smaller ions and molecules. This has, indeed, been shown to be the casein the cockroach nervous system (Treherne et al. 1973), in which the electricalresponses of urea-treated connectives showed greatly increased access of K+ andNa+, but no increased invasion of ionic lanthanum beyond the perineurium wasobserved.

It is difficult to establish whether the functional tight junctions are those betweenthe basal borders of adjacent perineurial cells, or whether they are between peri-neurial and underlying glial cells; this difficulty is due to the impossibility in manycases of deciding whether a cell process lying near the surface, but not directly nextto the neural lamella, is perineurial or glial in nature. Certainly there appear to bezonulae occludentes between glia and some evidence has been presented here tosuggest that it is these which prevent lanthanum from migrating throughout thewhole nervous system when it has entered at one focal point. This evidence arisesboth from observations on the neurohaemal regions and on the tissues incubated inlanthanum dissolved in calcium-free saline where the lanthanum does pervadebeyond the perineurium, but only into a limited area around the point of entry.Where the neurosecretory axons terminate under the neural lamella, it is clear that


they are completely accessible to substances in the extracellular environment. Further,it can be seen that the lanthanum does leak back up into the intercellular spacesbetween the neurosecretory endings and the surrounding perineurial and glial cells;it is between the glial cells adjacent to the neurosecretory axons that zonulae occludentesexist which appear to restrict further movement of the lanthanum ions.

It is obvious that the nervous system must have a mechanism whereby neuro-secretory hormonal products can be released into the haemolymph and it is moreconvenient that no restrictive sheath should be interposed at such release points.However, it is also clear that the haemolymph is not always a favourable environmentfor the nervous system, particularly in phytophagous insects, and so restriction toextensive entry of exogenous substances, as maintained by glial occluding junctions,would be a convenient solution to this problem. The fact that fewer neurosecretoryterminals have been seen in adult than in 5th instar Rhodnius may suggest thatfewer hormones and/or lesser amounts of hormones are released in the adult.

With tissues incubated in calcium-free saline containing ionic lanthanum, occasionalopenings in the blood-brain barrier of Rhodnius have been observed. This seems tooccur when the perineurium is particularly attenuated, so it may be that thereare fewer occluding junctions to be disrupted in these areas. This interpretation hassome support in the results of other workers, since altered permeability to lanthanumhas been considered elsewhere to indicate disrupted tight junctions (Neaves, 1973).Removal of calcium is known to cause disaggregation of cells (cf. Hays, Singer &Malamed, 1965) and so it is likely that lack of calcium leads to dissociation of celljunctions which would allow penetration of substances through a normally restrictedpassage. Beyond the level of the perineurium, as mentioned above, local inter-glialtight junctions may prevent more extensive pervasion of the nervous tissue byexogenous substances, although they would presumably not inhibit diffusion of verysmall molecules and ions. At any rate the results of incubating tissue in calcium-freesolutions indicate that the perineurial junctions are being 'opened' in a way whichremains physiological, since the lanthanum is present in low concentrations, incontrast with opening the barrier due to high concentrations of lanthanum. In thisconnexion it is rather surprising that EGTA-containing solutions do not lead todisruption of the perineurial barrier. However, since the calcium-free incubationleads only to the occasional opening, perhaps fortuitously encountered, possibly therelatively infrequent invasion beyond the perineurium produced by the EGTA andsubsequent incubation has simply not been discovered, given the small number ofsections examined relative to the total surface area of perineurium in all the nerves.

No physiological importance can be attributed to the results observed usinghigh concentrations of lanthanum. However, it is clear that incubation with 500 mMlanthanum can be employed as an experimental tool to 'open' the system should itbe useful to disrupt completely the perineurial barrier. It is difficult to explain howthese hypertonic solutions might operate in opening the junctional complexes, butthe lanthanum is probably a competitor with calcium for sites which mediate in theregion of close membrane apposition, and this, together with the osmotic shock tothe system, which undoubtedly causes shrinkage of the cells, could lead to the


opening of the perineurial barrier in the dramatic fashion demonstrated. Moreover,at these concentrations, the ionic lanthanum could well be partly colloidal. On theother hand, since a finite leak in the perineurial barrier has previously been postulated(Pichon, Moreton & Treherne, 1971), it is not possible to exclude the possibilitythat with 500 mM lanthanum, one is creating an enormous concentration gradientfrom the neural lamella to the base of the perineurial clefts (which contrasts withthe much smaller gradient produced with lower lanthanum concentrations when thetracer does not penetrate beyond the perineurium), so that this finite passive leakis actually being visualized with the invasion of lanthanum into the extracellularspaces beneath the perineurium.

In vertebrate systems, 240 mM urea applied to toad urinary bladder apparentlyincreased the permeability of the zonulae occludentes by producing discontinuitiesin the fibrils that are characteristic of the junctions when viewed by freeze-fracturingmethods (Wade & Karnovsky, 1974). Hypertonic concentrations (0-5 M) of suchdisaccharides as sucrose led to the splitting of both gap junctions and zonulaeoccludentes in mouse liver as monitored by morphological changes revealed byfreeze-cleave techniques (Goodenough & Gilula, 1974). Any structural alterationsin the junctions in rat Sertoli cells are observable only after freeze-fracture techniquesand are not evident in conventional sections, although new-found permeability tolanthanum indicates their altered nature (Neaves, 1973). In this connexion it isinteresting to recall that neither 0-5 M sucrose nor 3 M glucose has any effect onlanthanum uptake beyond the perineurial occluding junctions in Rhodnius or thecockroach respectively (Treherne et al. 1973); perhaps any junctional membranemodification induced by sucrose in insects is rapidly reversible. Further studiesusing freeze-cleaving techniques are obviously required to clarify the structure andincubation-induced modifications of perineurial junctions in insect tissues.

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the electrical activity of the cockroach abdominal nerve cord. J. exp. Biol. 39, 631-641.TREHERNE, J. E. (1965a). Some preliminary observations on the effects of cations on con-

duction processes in the abdominal nerve cord of the stick insect, Carausius morosus. J. exp.Biol. 42, 1-6.

TREHERNE, J. E. (1965 A). The distribution and exchange of inorganic ions in the centralnervous system of the stick insect, Carausius morosus. J. exp. Biol. 42, 7-27.

TREHERNE, J. E. (1974). The environment and function of insect nerve cells. In Insect Neuro-biology, Frontiers of Biology, vol. 35 (ed. J. E. Treherne), chapter 4. Amsterdam: North-Holland Publishing.

TREHERNE, J. E. & PICHON, Y. (1972). The insect blood-brain barrier. In Advances in InsectPhysiology, vol. 9 (ed. J. E. Treherne, M. J. Berridge & V. B. Wigglesworth), pp. 257-313.New York and London: Academic Press.

TREHERNE, J. E., SCHOFIELD, P. K. & LANE, N. J. (1973). Experimental disruption of theblood-brain barrier system in an insect (Periplaneta americana). J. exp. Biol. 59, 711-723.

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WIGGLESWORTH, V. B. (1959a). The histology of the nervous system of an insect RJiodmusprolixus (Hemiptera). I. The peripheral nervous system. Q.Jl microsc. Sci. ioo, 285-298.

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{Received 12 December 1974)


All the micrographs are taken from abdominal nerves of 5th instar or adultRhodnius prolixus.

Fig. 1. Transverse section of neurosecretory endings (ns) between perineurium (pn)and neural lamella (nl) of an unincubated control nerve, showing the typical dense-cored neurosecretory vesicles (dv). Note the absence of any cellular ensheathmenton the upper surface of the ending, a, glial-ensheathed axons; lid, hemidesmosomes.Arrow indicates the septate junction between adjacent perineurial cells, x 34000.Fig. 2. Cross-section of neurosecretory endings (ns) surrounded by dense depositsof lanthanum within the neural lamella (nl). The perineurial (pn) clefts are alsoinfiltrated with the tracer. Note the absence of lanthanum in the underlying extra-cellular space between glia (g) and axons (a). The preparation was incubated for60 min in a 5 mM lanthanum solution, x 34000.Fig. 3. Longitudinal section through 2 neurosecretory endings (ns) surrounded bylanthanum, the upper one being completely encompassed at this point by the neurallamella (nl). The lanthanum penetrates through the neural lamella to the intercellularspaces of the perineurium (pn) but not beyond. Note the longitudinal arrays ofmicrotubules in the glia (g) beneath the perineurium (see also Fig. 17). Materialincubated with 5 mM lanthanum for 60 min. x 30000.

Fig. 4. Cross-section through 2 neurosecretory endings (nsu ns«), one lying beneaththe perineurium (pn), within the axon (a) and glial mass. Note the lanthanumsurrounding this ending (ns2) and lying in an interglial cleft (arrow), although onlyin that adjacent to the actual ending, suggesting the existence of some restriction,possibly a junction, to further penetration, nl, neural lamella. Incubated for 2 h in5 mM lanthanum solution, x 35000.

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Figs. 5 and 7-12 are junctions between the perineurial cells of Rhodnius nerves.

Fig. 5. Complex interdigitations between adjacent perineurial cells where septatejunctions (sj) and possible continuous junctions (c) occur. The axons (a) are en-sheathed by glia (g) which contain microtubular arrays. Control preparation, incubatedin lanthanum-free saline for 120 min. x 60000.

Fig. 6. Extracellular space (s) between attenuated perineurial process (pn) andunderlying glial cell (jg). The membrane of the latter exhibits the density (arrow)characteristic of a hemidesmosome. nl, neural lamella, x 153000.

Fig. 7. Zonulae occludentes (arrows) between adjacent perineurial cells. A, x 75000;B, x 110000.

Fig. 8. Lanthanum penetrating the clefts between perineurial (pn) cells butstopping before entering a dilated extracellular space. Micrographs of this sortsuggest that it may be a zonula occludens (arrow) which restricts further penetrationof the tracer. The underlying glial cells (g) are joined by a gap junction (gj). Incubatedfor 60 min in 5 mM lanthanum, x 92000.

Fig. 9. Perineurial gap junction just beneath the neural lamella (nl). Note itsnarrow intercellular cleft (arrow) in contrast with the normal space (s). x 88000.

Fig. 10. Gap junction (arrow) between perineurial cells, filled with lanthanum afterincubation in a 5 mM solution for 60 min. c, normal intercellular cleft, x 105000.

Fig. 11. Septate junction (sj), gap junctions (gj) and zonula occludens (arrow)between interdigitating perineurial cells, x 128000.

Fig. 12. Lanthanum penetrating intercellular perineurial spaces in a region ofseptate junctions (sj); the lanthanum deposits can be seen to disappear abruptly(arrow), possibly at a zonula occludens. Incubated in 5 mM lanthanum for 60 min.x 76000.

Neuroliaemal regions in insect nerves 193

194 N. J. Lane, R. A. Leslie and L. S. Sioales

Fig. 13. Neurosecretory terminals (us) lying partly in the neural lamella (nl) sur-rounded by lanthanum. The perineurium (pti) is several thin layers thick and underit lie the axons (a) and glia (g). The tissue was incubated in Ca!+-free saline containing5 mM LaClj for 60 min. Note that the tracer (arrows) has not penetrated beyondthe level of the perineurium. fb, fat body, x 43000.Fig. 14. Tissue incubated, as in Fig. 13, in Ca1+-free saline containing 5 mM LaCl3,for 60 min. In this region of the nerve the perineurium (pn) is extremely attenuatedand the lanthanum has penetrated to the level of the axon (a) and glial (g) mass,where the axons are completely surrounded by the lanthanum. Note, however, thatthe lanthanum does not pervade the whole of the intercellular space, and the insetshows the sort of structures found (arrow) which suggest that tight junctions maybe stopping the further invasion of the lanthanum, fb, fat body; nl, neural lamella,x 49000; inset, x i 12000.

Fig. 15. This material has been pre-incubated in 0-5 M sucrose for 2 min followedby treatment in 10 nui LaCl3 for 30 min. Note that there is intercellular perineurialpenetration by the tracer and the neurosecretory ending (ns) is surrounded by it butthe 'barrier' is still intact in that lanthanum does not pervade the underlying inter-glial (g) and axonal (a) spaces, nl, neural lamella, part of which is extending into theperineurium (pn). The tracheole (^-containing cell is also encompassed by lanthanum,x 54000.Fig. 16. Incubation in 100 mM LaCl, for 60 min shows that although the perineurium(pn) is penetrated with tracer, the underlying extracellular spaces and axon (a) andglial (g) surfaces are free of lanthanum, nl, neural lamella, x 51 000.


Fig. 17. Axons (a) surrounded by glial cells (g) showing the highly ordered arrays ofmicrotubules in the glial cytoplasm, x 49000. Inset shows a sonula occlndens (tightjunction) between 2 glial cells (arrow), x 128000.Fig. 18. Tissue incubated for 1 h in 500 mM lanthanum. The 'barrier' has beendisrupted and there is a total tracer penetration (arrows) throughout the nervebeyond the perineunum to the level of the axon (a) surfaces. Note the regular arraysof microtubules within the glial (g) cytoplasm, x 55000.Fig. 19. Nerve incubated as in Fig. 18 whereby the lanthanum penetrates to thelevel of the glial-axonal (a) mass. Note a number of microtubules in the glial cellswith lanthanum-filled interiors (arrows). This may reflect unusual relationshipsbetween the microtubules and the plasmalemma immediately beside the lanthanum-filled extracellular spaces (s). x 93 000.


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