J. Cell Sci. 41, 341-368 (1980) 341Printed in Great Britain © Company of Biologists Limited 1980

CELL JUNCTIONS IN THE EXCITABLE

EPITHELIUM OF BIOLUMINESCENT SCALES

ON A POLYNOID WORM: A FREEZE-FRACTURE

AND ELECTROPHYSIOLOGICAL STUDY

A. BILBAUTLaboratoire d'Histologie et Biologie Tissulaire, University Claude Bernard, 43 Bd. du11 Novembre 1918 69621, ViUeurbairne, France.

SUMMARY

The bioluminescent scales of the polynoid worm Acholoe are covered by a dorsal and ventralmonolayer of epithelium. The luminous activity is intracellular and arises from the ventralepithelial cells, which are modified as photocytes. Photogenic and non-photogenic epithelial cellshave been examined with regard to intercellular junctions and electrophysiological properties.Desmosomes, septate and gap junctions are described between all the epithelial cells. Lan-thanum impregnation and freeze-fracture reveal that the septate junctions belong to the pleated-type found in molluscs, arthropods and other annelid tissues. Freeze-fractured gap junctionsshow polygonal arrays of membrane particles on the P face and complementary pits on the Eface. Gap junctions are of the P type as reported in vertebrate, mollusc and some annelid tissues,d.c. pulses injected intracellularly into an epithelial cell are recorded in neighbouring cells.Intracellular current passage also induces propagated non-overshooting action potentials in allthe epithelial cells; in photocytes, an increase of injected current elicits another response whichis a propagated 2-component overshooting action potential correlated with luminous activity.

This study shows the coexistence of septate and gap junctions in a conducting and excitableinvertebrate epithelium. The results are discussed in relation to the functional roles of inter-cellular junctions in invertebrate epithelia. It is concluded that the gap junctions found in thisexcitable epithelium represent the structural sites of the cell-to-cell propagation of actionpotentials.

INTRODUCTION

Three classical types of intercellular junctions are generally described in inverte-brate epithelia. These are desmosomes, septate and gap junctions (Staehelin, 1974).Desmosomes and gap junctions can be demonstrated in a large variety of invertebrateand vertebrate tissues (Staehelin, 1974; Larsen, 1977) while septate junctions haverarely been reported in vertebrate tissues (Connell, 1978). Electrophysiologicalinvestigations, using intracellular recording and tracer injection, demonstrated ionicand metabolic coupling between non-excitable adjacent cells in invertebrate (Loewen-stein & Kano, 1964; Loewenstein, 1976) and vertebrate (Petersen, 1976; Hammer &Sheridan, 1978) glandular epithelia. The cell-to-cell propagation of action potentialshas been described in invertebrate and vertebrate excitable tissues such as epithelia(Mackie, 1965, 1976; Roberts & Stirling, 1971; Kater, Rued & Murphy, 1978),cardiac and smooth muscle (Barr, Dewey & Berger, 1965; Barr, Berger & Dewey,

342 A. Bilbaut

1968) and nervous electrical synapses (Watanabe & Grundfest, 1961; Bennett, Naka-jima & Pappas, 1967). Cell coupling in both non-excitable and excitable tissuesinvolves low-resistance pathways which have been correlated, in many instances, withthe presence of gap junctions (Bennett, 1978). In invertebrate epithelia there is nodirect evidence that the gap junctions act as coupling sites or are the only couplingsites (Satir & Gilula, 1973; Staehelin, 1974; Bennett, 1978); this will be discussed ina later section.

The object of the present study is to characterize intercellular junctional structuresfound in an invertebrate epithelium which has conducting, excitable and biolumine-scent properties. The polynoid worm Acholoe astericola (dell Ch) displays strongluminous activities after electrical or mechanical stimulation. Spontaneous lightemission has never been observed in Acholoe and the significance of the biolumine-scence in all the polynoid worms studied remains purely speculative (Haswell, 1882;Nicol, 1953). In Acholoe, luminous activities originate in the scales (elytra) which arethin, epithelial diskoidal plates arranged dorsally in a double row. Each is attached toa segment by a short peduncle and consists of a simple-layered continuous epitheliumcovered by a collagenous cuticle. On the ventral face, the epithelial cells are differen-tiated as photocytes which constitute a homogeneous cell population, the photogenicarea. Photocytes contain several granules of paracrystalline endoplasmic reticulum(Bassot, 1966) which are the intracellular sources of the bioluminescent activity of thescales (Bassot & Bilbaut, 1977a). Pavans de Ceccatty, Bassot, Bilbaut & Nicolas (1977)reported that the 2 epithelial planes delimit an internal compartment crossed bymultiple cellular processes arranged perpendicularly on both epithelial faces. Theseprocesses originate from the basal pole of all the epithelial cells and also from a specialcell type found in the internal compartment, the 'clear cells'. All the processes containbundles of filaments. Nerve trunks arise from a ganglion near to the centre of thescale and end at numerous peripheral sensory cells.

An extracellular electrical pulse applied to the photogenic area of an isolated scaleproduces at least one brief flash (60-120 ms in duration). During repetitive electricalstimulation (1 pulse/s), the intensity of successive flashes at first increases rapidly,for a short period, then decreases slowly (Bilbaut & Bassot, 1977). Autophotographicobservations after image intensification of the photogenic area show several strikingfeatures (Bassot & Bilbaut, 19776). Firstly, during one flash, the illumination startsat the stimulated site and spreads towards the periphery of the luminous effector.Secondly, at the beginning of a repetitively stimulated light emission, only a part of thephotogenic area is illuminated. This active zone is initially limited to an area close tothe stimulated site and, flash by flash, increases gradually in extent. When the flashintensities reach their maximum value, the whole of the photogenic area is illumin-ated. The slow decline of the luminous intensities of flashes results from the progressiveexhaustion of bioluminescent products. No recovery of bioluminescent capacitiesoccurs in isolated scales (Bilbaut & Bassot, 1977).

Luminescent propagation in the photogenic area could result from the presence ofeither a multi-innervated uncoupled epithelial cell system or a conducting epithelialcell system. Ultrastructural observations (Pavans de Ceccatty et al. 1977) did not

Cell junctions in excitable epithelium 343

demonstrate terminal neuro-effector junctions ending on photocytes, whereas numerouscell junctions, both septate and gap junctions, were found between all epithelial cells,supporting the possibility of a conducting epithelial system.

Cell coupling and cell excitability have been reported in photogenic and non-photogenic epithelial cells in Acholoe (Bilbaut, 1978 a) in a preliminary account andHerrera (1977, 1979) has also reported action potential propagation in the biolumines-cent scale epithelium of the polynoid worm Hesperonoe. In the present paper, themorphology of all the cell junctions found in the scale epithelium is detailed in thefirst section, using intercellular tracer and freeze-fracture techniques. In the secondsection, electrical coupling and excitability of epithelial cells are examined usingintracellular recording and stimulation techniques.

MATERIALS AND METHODSAcholoe astericola is a commensal of the starfish Astropecten aurantiacus. The starfishes were

collected by scuba-diving near the Marine Station of Banyuls-sur-Mer and transported inisothermic boxes to Lyon. They were maintained in natural seawater at a constant temperature(15 °C) and fed monthly with clams.

In order to avoid damage to the annelid during the sampling of the scales, worms werecarefully removed from the starfish, transferred to a Petri dish filled with seawater and placedin a freezer. When the first ice-crystals appeared in the seawater, worms were immobilized fora few minutes and individual scales were cut off using fine scissors.

Scanning electron microscopy

Scales were fixed by immersion for 60 min at room temperature in 3 % glutaraldehydein o-i M Na cacodylate-HCl buffer, pH 7-4, containing 0-3 M NaCl. The cuticle covering thescale was carefully peeled off on one of the epithelial faces in the washing solution (0-3 MNaCl in 02 M Na cacodylate-HCl buffer at pH 7'4). Cuticle pieces with adhering epitheliumwere dehydrated in acetone and critical-point dried (acetone-CO| substitution). Observationsof preparations were made with a Cambridge (S600) scanning electron microscope.

Transmission electron microscopy

For conventional fixation, the scales were cut into small pieces, fixed and washed for 1 haccording to the above procedure, postfixed for 1 h at room temperature in 1 % osmiumtetroxide and 0-4 M NaCl buffered by o-i M Na cacodylate-HCl at pH 7-4, dehydrated andembedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate beforeexamination.

The lanthanum solution was prepared using Revel & Karnovsky's method (1967); fixed andrinsed specimens were postfixed for 4 h at room temperature. After dehydration and embedding,thin sections were lightly stained with lead citrate.

Freeze-fracturing

Whole scales were fixed for 1 h in glutaraldehyde solution, washed for 30 min in NaClbuffered solution then immersed in successive baths of 10 and 20 % glycerol solution for 20 minand for 3 h in 30 % glycerol solution.

Several scales were placed on gold supports, rapidly frozen in liquid freon 22 (—160 °C) andtransferred to liquid nitrogen until used. Freeze-etching was carried out in a Balzers B.A.F.300 device. The specimens were cleaved at -100 °C and etched for 1 min at a vacuum of1-2 x io~* torr (1-33 — 2-66 x io~4 N m"1). The exposed surface of the specimen was shadowedat an angle of 450 with platinum/carbon. Replicas were made by evaporating carbon onto the

344 A. Bilbaut

shadowed surface. The thickness of shadowing (2 run) and of the replica (206 nm) werechecked with a quartz crystal thin film monitor. The replicas were cleaned overnight with 20 %chromic acid.

The direction of shadowing in the micrographs is shown by encircled arrows. Fracture-facenomenclature is that of Branton et al. (1975). Replicas and thin sections were examined with aHitachi HU 12A or Philips EM 300 electron microscope.

Electrophysiology

The presence of cuticle on the external face of the epithelial cells prevented penetration ofmicroelectrodes into the cells. For this reason, scales were cut into narrow strips, transferredto an experimental dish where they were maintained in a vertical position by 2 suction elec-trodes arranged on each face of the preparation (Fig. 1). Under these conditions, the 2 epi-thelial layers were visible with a binocular microscope and were directly accessible to themicroelectrodes.

Fig. 1. Schematic drawing of the experimental set-up. Scale fragment (sf) is in themiddle of the figure; dark and dotted lines limit the surface of the photogenic area(pa) on the lower epithelial layer (le). Other explanations in the text. Abbreviations,c: commutator; dm: double-barrelled microelectrode; m: microelectrode; pm: photo-multiplier tube; r: recording; re: reference electrode wire; se: suction electrode; si:stimulus isolation unit; ue: upper epithelial layer.

The suction electrodes utilized for extracellular stimulation were made from drawn poly-ethylene tubing containing chloride silver wire connected to a stimulus isolation unit; squarepulses could be delivered from either suction electrode. Simple and double-barrelled glassmicroelectrodes were pulled on a solenoid microelectrode puller (Narishige, PD5) and filledwith 3 M KC1; they had an initial resistance of 40-60 MCI. Square pulses of stimulation current,monitored by another stimulus isolation unit, were injected intracellularly by one half of thedouble-barrelled microelectrode, the other recording continuously the membrane potentialof the impaled epithelial cell.

The size of epithelial cell bodies is around 15-20 /tm and in order to obtain coupling recordsbetween epithelial cells, a simple microelectrode was inserted in another cell 100-200 fim awayfrom the current-passing electrode in the same epithelial layer (Fig. 2). The 2 recording micro-electrodes were connected to the input of a high-impedance preamplifier (Fig. 1). Electricalsignals were displayed on an oscilloscope (Tektronix, 5103N) and an oscillographic recorder(Hewlett Packard, 7402A).

Luminous activity was picked up by one extremity of an optic fibre adjusted over the pre-

Cell junctions in excitable epithelium 345paration, the other being connected to a photomultiplier tube (R.C.A. 1P21). Anode currentwas displayed on the oscilloscope and oscillograph recorder.

The saline solution had the following composition: NaCl, 500 nrn; KC1, 10 HIM; CaCl.,20 mM; MgClj., 12 HIM; tris (hydroxymethylarninomethane), o-oi M; pH of the solution wasadjusted to 7-4 with HC1.

All experiments were performed at room temperature (20 °C).

RESULTS

Desmosomes

The epithelial cells seen in thin section have well developed belt desmosomes(zonula adherens). They surround the top of the epithelial cells close to the cuticle-epithelium interface (Fig. 3). The intercellular space, 15-17 nm wide, is filled withfibrous material. The protoplasmic leaflets of the cell membranes are lined withdense material from which bundles of 5-7 filaments emerge at places (Fig. 4). Theextremity of the intercellular cleft opens into a vesicular system below the cuticle.

Fig. 2. Transverse fracture of scale fragment as seen by scanning electron microscopy.Upper and lower epithelial planes (arrows) correspond respectively to the photogenicand non-photogenic epithelium. These 2 opposite epithelial layers delimit an internalcompartment (ic) crossed by numerous cell processes (cp) arising from all epithelialcells. The lower epithelium lies on the cuticle (large arrow) which was removed fromthe upper epithelial plane during preparation of the specimen. For electrophysiologicalexperiments, the 2 microelectrodes were inserted in either epithelial plane, x 950.

Spot desmosomes (macula adherens) are observed in the depth of the epitheliallayer. They are principally differentiated between the bodies of epithelial cells andthe filament-containing processes which arise from the opposite epithelial face (Fig. 2)or from the 'clear cells' of the internal compartment. They are sometimes foundbetween 2 cell processes and more rarely between the bodies of adjacent epithelialcells. They are the most abundant cell junctions of the scale epithelium (Fig. 5). Theintercellular space about 14-15 nm wide is filled with a cementing material; a denselamina lies in the middle of the junctional cleft. Another dense plaque is closelyattached to the cytoplasmic leaflet of the cell membrane. Filaments around 8-10 nm

23 CEL 40

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Cell junctions in excitable epithelium 347

in diameter are generally associated with both cytoplasmic dense plaques (Fig. 6).These filaments have the same diameter as those found in the cell processes. Often,spot desmosomes appear to have an asymmetrical aspect. This results from the lowerelectron density of one of the cytoplasmic plaques from which arise io-nm filaments(Fig. 1).

Septate junctions

Septate junctions occur between all the epithelial cells just below the belt des-mosomes (Fig. 3). They are generally found in an area of membrane interdigitation.In transverse sections of conventionally stained preparations, the intercellular space,15-16 nm in width, is crossed by electron-dense septa about 10 nm thick, which jointhe membranes of 2 adjacent cells (Fig. 8 A). The centre-to-centre spacing of thesepta is 17-18 nm. Tangential sections of the junctional area reveal a honeycomb-likestructure in the intercellular space (Fig. 8 B).

Colloidal lanthanum infiltrated imperfectly and discontinuously through the inter-cellular spaces of the epithelial cells; however, lanthanum accumulation was oftenpronounced in the septate junctions. In transverse sections, electron-lucent septa,6-7-5 nm thick, were finely delineated by electron-dense deposits of lanthanumfilling the remaining intercellular space (Fig. 9). Septal periodicity is about 17 nmcentre-to-centre. Septate junctions with regular and clear-cut septa are infrequentlyobserved in the epithelium; in most cases, septa are widely spaced (Fig. 10) and oftenthe intercellular cleft of 16-17 nm width appears devoid of septa; it may enclosedisorganized electron-lucent material that lanthanum reveals occasionally. In obliquesections, lanthanum-infiltrated septate junctions display transparent, pleated bandslying in the intercellular cleft (Figs. 11, 12). The periodicity of pleating is about 23 nm.In some cases, electron-lucent pegs emerge at the apex of the pleats (Fig. 12). The

Fig. 3. Thin, section through the non-photogenic epithelium showing the apical poleof cells covered by the cuticle (c). Several cell junctions are seen between the 2epithelial cells (C1 and Ca). They are desmosorr.es (d), septate (j;) and gap junctions(gj). x 52coo.Fig. 4. Belt desmosome as seen in transverse thin section of the non-photogenicepithelium. The intercellular cleft is filled with fibrous material. The electron-denselamina (/) is against the protoplasmic leaflet of the cell membrane. Fibrillar (/) com-ponents emerge at places, x 56500.Fig. 5. Tangential thin section in the photogenic epithelium showing a photocyte (p)with nucleus (n), and several granules of paracrystalline endoplasmic reticulum, thephotogenic granules (pg). As shown by the large extracellular spaces {es) lined bynumerous spot desmosomes (sd), this section passes through the basal regions ofepithelial cell bodies where the cell processes containing filaments (/) begin to indi-vidualize, x 15500.Fig. 6. Enlargement of spot desmosomes. Note the lamina (arrow) in the middle of theintercellular cleft and 8-10 filaments (/) lying parallel to the electron-dense plaqueagainst the inner layer of the cell membrane, x 65000.Fig. 7. Enlargement of the region outlined in Fig. 5, showing the asymmetricalappearance of spot desmosomes. On one of the junctional faces, cytoplasmic plaques(arrows) appear less contrasted and the filaments (/) applied against them are morescattered than on the opposite junctional faces; es, extracellular space, x 39000.

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Cell junctions in excitable epithelium 349

bands are variable in disposition; they can be arranged in straight (Fig. 11) or looped(Fig. 12) parallel arrays and even within a parallel pattern, some bands exhibitdirectional change (Figs. 11, 12).

Freeze-fractured septate junctions reveal rows of intramembranous particlesassociated with the P face (Fig. 13) and complementary furrows associated with theE face (Figs. 13, 14). Junctional membrane particles are irregularly spaced in the rows;some are isolated while others are packed together and appear fused over a shortlength (Fig. 13). The diameter of the isolated junctional particles is 9-15 +0-9 nm(n = 133). The rows of junctional particles show various configurations on thejunctional surface. Generally, they constitute straight or curved parallel arrangements;the space between rows varies between 10 and 25 nm. Favourable freeze-fracturereplicas sometimes exhibit extensive junctional surfaces characterized by a discon-tinuity in the parallel patterns of rows of membrane particles (Fig. 15); at the peri-phery of parallel arrangements some rows of particles diverge and run along the wholejunctional surface in undulating or looped configurations; occasionally, they may endabruptly but more often enter new parallel patterns with other rows of membraneparticles (Fig. 15).

Some rows of junctional particles are very short and both extremities are observedin the same junctional surface (arrowheads, Fig. 15). Infrequently, a row of junctionalparticles is seen to converge towards another (asterisks, Fig. 15).

In freeze-fractured septate junctions, the E face often shows long sinuous membranedepressions lined by one row of membrane particles (Fig. 14). On the P face,complementary ridges enclose some membrane particles and holes (Fig. 15).

Fig. 8. Septate junctions as seen in conventional thin section, A, transverse sectionof a septate junction in the non-photogenic epithelium. Distinct and regularly spacedsepta (arrows) are shown, x 73000. B, septate junction in oblique section. Note thehoneycomb-like pattern lying in the intercellular cleft. Photogenic epithelium,x 96000.Figs. 9-12. Septate junctions revealed after lanthanum impregnation of the inter-cellular spaces.

Fig. 9. Section perpendicular to plasma membranes showing transparent regularlyspaced septa (arrows) crossing the intercellular cleft. Non-photogenic epithelium,x 80000.

Fig. 10. In this section perpendicular to plasma membranes, transparent septa(arrows) are irregularly spaced in the intercellular cleft. Photogenic epithelium,x 79000.

Fig. 11. Section oblique to the plasma membranes reveals transparent pleatedbands (arrows) lying in the intercellular cleft; in this picture, pleated bands arearranged in a parallel straight pattern. The pleated band above the upper arrowshows a change in direction. Photogenic epithelium, x 127000.

Fig. 12. Another configuration of pleated bands in a parallel looped pattern.Note changes in direction of some pleated bands (arrows) which display pegs at theapex of the pleats. Non-photogenic epithelium, x 150000.

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Cell junctions in excitable epithelium 351

Gap junctions

Numerous gap junctions are found between all the epithelial cells, in the non-photogenic epithelium (Fig. 16) as well as in the photogenic epithelium (Fig. 17). Intransverse sections of conventionally stained preparations, the gap junctions have aseptalaminar configuration. The intercellular space is about 2-5-2 nm wide. Gapjunctions 0-2-3 M™ m length are generally arranged in clusters below the septatejunctions. Infrequently, they may be observed in the septate junctional region(Fig. 18). Lanthanum-filled gap junctions in transverse section show a pentalaminarconfiguration (Fig. 18), while tangential sections reveal a structural organization in theintercellular space of subunits (Fig. 19). These subunits assembled in polygonalpatterns are 11-12 nm apart (centre-to-centre) and sometimes have a central densespot (Fig. 19).

The existence of gap junctions in the scale epithelium is confirmed by examinationof freeze-fracture replicas. Gap junctions appear as small diskoid assemblages,between 0-08 and 0-3 /*m in diameter, of intramembranous particles associated withthe P face (Fig. 20) and a lattice of pits associated with the E face (Fig. 21). Junctionalmembrane particles are irregularly arranged on the P face in polygonal arrays and aresometimes closely packed; the size of isolated junctional particles is 11-7+1-211111(n = 71). In some cases, a 2-nm dot can be detected in the centre of junctional particles(Fig. 20). On the P face there is an area of membrane surrounding each junctionalplaque that is sparsely populated with non-junctional membrane particles (Figs. 20,22). On the E face, the pits are often regularly arranged in a hexagonal lattice;their density is 7000-8000 per /*m2 (Fig. 21).

Fracturing patterns of gap junctions are varied. Isolated junctional particles may beobserved on the E face (Fig. 21). In other cases, E face junctional fragments are

Figs. 13-15. Freeze-fractured preparations of prefixed and glycerinated scale epi-thelium showing fracturing profiles of septate junctions.

Fig. 13. Several rows of junctional intramembranous particles are associated withP face (PF) and complementary furrows are seen on the E face (EF). The white linebetween P and E face is the fractured intercellular space (ie). On the P face, somejunctional particles are closely packed (arrows). Photogenic epithelium, x 131000.

Fig. 14. Septate junction showing fine parallel furrows on the E face (EF). Thelong sinuous membrane depression lined by one row of membrane particles (arrows)corresponds to an intersection between 3 cells. At bottom right, a P face (PF) frag-ment is seen. Non-photogenic epithelium, x 77800.

Fig. 15. Extensive fracturing pattern of a septate junction revealed on a P face (PF).Three parallel arrangements of rows of membrane particles (1, 2, 3) are seen. Betweenparallel arrangements, rows of junctional particles show various configurations with,sometimes, 'anastomosing sites' (asterisks). Several rows of membrane particles endin the junctional surface (arrowheads). In the lower middle of the picture, thetransverse ridge (large arrowheads) on which some particles and holes are seen,probably corresponds to the line of intersection between 3 cells. E face fragments (EF)in the upper left and bottom right remained associated with the P face. Non-photo-genic epithelium, x 101 800.

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Cell junctions in excitable epithelium 353

removed during fracturing, revealing junctional particles associated with the sub-jacent P face of the opposite cell membrane (Fig. 21). Frequently, E face junctionalfragments remain associated on the complementary P face sometimes with a clearpolarity as shown in Fig. 22.

In many instances, gap junctions revealed by freeze-fracturing appear in clusterscontaining as many as 30 junctional areas (Fig. 22); they have not been observed inseptate junctions but only near them (Fig. 23).

Membrane potential

Microelectrode penetration into both types of epithelial cells produces an abruptnegative change in potential which passes from the reference value - zero potential -to values around — 70 mV. The mean membrane potential measured in 72 differentpreparations (one cell per preparation) is — 717 + 5-5 mV. Usually, this resting potentialremains steady when the epithelium is not stimulated.

Action potentials evoked in epithelial cells by extracellular stimulation

When an extracellular electrical pulse is applied on either face of the scale epithelium,all-or-none action potentials are observed by intracellular recording in both photogenicand non-photogenic epithelial cells.

Photocytes display 2 separate all-or-none responses which are stimulus-strengthdependent. The first, which results from weak stimulation pulses (5-7 V during 10ms),is a simple non-overshooting action potential (Fig. 24A). The second, generated by

Fig. 16. Transverse section, of a conventional preparation showing 2 gap junctions(arrows) below a pigment granule (pg) in the non-photogenic epithelium, x 64500.

Fig. 17. Three gap junctions (arrows) close to a photogenic granule (phg) are seen in atransverse thin section of a conventional preparation, x 64000.

Fig. 18. Pentalaminar configuration of a gap junction (arrow) revealed by lanthanumtreatment of the preparation. This gap junction is enclosed in a septate junction (sf);pg, pigment granule. Non-photogenic epithelium, x 72000.

Fig. 19. Oblique section of a lanthanum-infiltrated gap junction showing sub-unitsarranged in polygonal patterns in the intercellular cleft. Note the central pore in somesub-units of the junctional regions circled by dark lines. Non-photogenic epithelium,x 94300.

Figs. 20-23. Freeze-fractured preparations of prefixed and glycerinated scale epi-thelium showing fracturing profiles of gap junctions.

Fig. 20. On a P face (PF), gap junctions consist of macular arrays of intramembra-nous particles closely packed in irregular arrangements. A central dot can be seen insome junctional particles (circles). Note the ring with few non-junctional membraneparticles surrounding the junctional area. In the bottom left of the micrograph,fractured intercellular space and E face (arrow) are seen. Numerous non-junctionalmembrane particles are scattered on the P face. Non-photogenic epithelium, x 138 000.

Fig. 21. On an E face (EF), gap junctions correspond to a macular lattice of pitsmore or less hexagonally arranged. Some junctional particles remain associated withthe E face of the gap junction at the top. In the bottom, arrays of junctional particlesare associated with the P face of the opposite cell; the clear band at right (arrow) of thejunction is the fractured intercellular space. Photogenic epithelium, x 185000.

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Cell junctions in excitable epithelium 355

stronger stimulation pulses (7-12 V during 10 ms) is a 2-component action potentialbeginning with an initial overshooting fast spike followed by a delayed slow depolari-zation (Fig. 24B). Table i, which summarizes the wave-form characteristics of these2 action potentials, shows that only the amplitude and duration of the overshootingspike are at all constant.

In non-photogenic epithelial cells, extracellular stimulation pulses (5-7 V during10 ms) produce simple action potentials (Fig. 25). Their amplitude of 60-9 + 8 mV(n = 48) is lower than the zero potential level but weak overshoots (2-5 mV) haveoccasionally been observed. The duration measured at one-half amplitude is 41 -9 +13-2 ms (n = 48).

With extracellular stimulation, the delay in response is longer as the distancebetween both the stimulation and recording electrodes increases.

Luminous activity is specifically correlated with the 2-component overshootingaction potential (Fig. 24 B). Light intensity is stimulus-strength independent andvaries from one scale to another according to its 'load' of bioluminescent products;when these are exhausted, 2-component action potentials occur without luminousactivity. In a few cases, the simple non-overshooting action potential of photocyteswas accompanied by a very weak luminescence.

Electrical coupling of the epithelial cells

If a simple microelectrode is inserted in a photocyte close to the double-barrelledmicroelectrode inserted in another photocyte, a weak intracellular current injectioninduces a potential change in both cells (Fig. 26). According to the polarity of thecurrent passed in the cell, depolarizing and hyperpolarizing membrane potentialchanges are obtained (Fig. 31). Owing to technical limitations arising from intracellularstimulation with a double-barrelled microelectrode, membrane rectification propertieshave not been studied. The input resistance of the photocytes is low; some measure-ments give values around 1 MO. The amplitude of the potential change depends onthe interval separating the 2 microelectrodes; for a given value of current injection,this amplitude decreases as the distance between the microelectrodes increases.Coupling coefficients have not been measured, photocytes being too small to permitpenetration into two adjacent cells.

In the non-photogenic epithelium, similar observations also indicate the existenceof electrical coupling between these epithelial cell types (Fig. 27).

Fig. 22. Cluster of P-type gap junctions lying on a P face (PF). On most gapjunctions, junctional fragments of E face (EF) oriented towards the top of themicrogTaph remain associated with the P face. The white line limiting the upperpart of the gap junctions corresponds to fractured extracellular spaces. At the top left,an E face (EF) fragment is seen. At the bottom, cross-fractured membranes of 2adjacent cells (Cl and Ct) enclose 3 gap junctions (asterisks); note the narrowing of theintercellular cleft close to gap junctions (arrows). A cross-fracture of nuclear membraneshowing a pore (/>) is seen in the lower part of the micrograph. Photogenic epithelium,x 77000.

Fig. 23. P-type gap junctions (gf) near to a septate junction (sf) revealed on aP face (PF). Non-photogenic epithelium, x 161 500.

356 A. Bilbaut

Table 1. Summary of the waveform characteristics of action potentialsobserved in photogenic epithelial cells

Two component overshooting action potential (n = 50)*Simple non-overshooting , * 1action potential (n = 34)* Amplitude Durationf

Amplitude Durationf Spike Overshoot Second com- Spike Secondponent component

33-4 74-i 88-8 16-9 22-3 177 295±8-3 mV ±317 mV ±5'6mV ± 5 mV ±5-4mV ± 3 ms ± 135-8 ms

• n is the number of tested preparations (one cell per preparation) in 16 different animals,•f Duration were measured at one-half amplitude.

24A 24B 25

Fig. 24. Action potentials induced in a photocyte by extracellular electrical pulsesapplied to the photogenic epithelial layer. Fig. 24A: Simple non-overshooting actionpotential (lower trace); no luminous activity was recorded (upper trace); the electricalpulse (asterisk) was 7 V during 10 ms. Fig. 24B: In the same photocyte, increase ofthe stimulus (9 V during 10 ms; asterisk) evokes another action potential which is a2-component overshooting action potential (lower trace) correlated with an intenseluminous response (upper trace). In both figures, dashed lines represent zero potential.Fig. 25. Simple non-overshooting action potential (lower trace) induced in a commonepithelial cell by one extracellular pulse (6 V during 10 ms; asterisk) applied to thenon-photogenic epithelial layer. No luminescence was observed (upper trace). Dashedline represents zero potential.

Cell excitability and action potential propagation

Active responses appear in the intracellularly stimulated photocytes when theamounts of injected current during 200 or 300 ms are between io~7 and 2 x io~7 A.In all tested cells, membrane depolarization must be raised between — 20 and — 10 mVby intracellular current injection in order to elicit the spike. As a consequence of thisraised threshold, the waveform of the 2 responses produced in stimulated photocytesis difficult to determine. The first response is a weak positive deflection (Fig. 28 A) andis sometimes accompanied by a slight luminous activity. The second response whichis generated at a more positive threshold than the preceding one, is a large signalcorrelated with an intense flash (Fig. 28B). When a photocyte is stimulated by suc-cessive current injection (1 pulse/s), the luminous intensity increases in most cases

Cell junctions in excitable epithelium 357

until the third or fourth flash, then decreases. Repetitive electrical firing and luminousactivity also occur during intracellular current injection (Fig. 29); the luminousintensity of flashes is comparable whether the preparation is intra- or extracellularlystimulated (Fig. 30).

_ r

Jf.

JI50 ms

I27

Fig. 26. Demonstration of electrical coupling between 2 non-adjacent photocytes.Depolarizing current pulses were intracellularly injected into the cell by one branchof the double-barrelled microelectrode; membrane potential changes were recordedin the same cell by the other branch (middle trace). The lower trace is the propagatedvoltage deflection recorded by the simple microelectrode inserted in another neigh-bouring photocyte.The upper trace is the current monitor. Microelectrodes were about80 fim apart.Fig. 27. Demonstration of electrical coupling between 2 non-adjacent commonepithelial cells. Weak intracellular depolarizing current pulses injected in an epithelialcell induce membrane potential changes (middle trace) propagated with decrement inanother neighbouring cell (lower trace). The upper trace is the current monitor.Microelectrodes were about 80 fim apart.

In fragmented scales, the responses evoked in one photocyte by intracellularstimulation are propagated. If a second microelectrode is inserted in any otherphotocyte, both simple non-overshooting and 2-component overshooting actionpotentials (Fig. 31) are recorded, which correspond respectively to the first and secondresponses observed in the stimulated photocyte.

Similar results have been obtained on the non-photogenic epithelium whereexcitability and propagation of simple non-overshooting action potentials are observedduring intracellular current passage in an epithelial cell and intracellular recordingin another (Fig. 32). In these conditions, no luminescence is associated with theseaction potentials.

Further investigations show that action potentials elicited by intracellular currentinjection in one epithelial cell propagate from one epithelial layer towards the other.These results will be reported in a separate note which will detail correlations betweenaction-potential propagation in the epithelium of isolated scales and luminous activitiesobserved in the photogenic area.

358 A. Bilbaut

J]50 ms

2 8 A

Fig. 28. Action potential induced in a photocyte by intracellular current injectionand recording through a double-barrelled microelectrode. Fig. 28 A: the bottom traceis the first response to an intracellular depolarizing current pulse of 250 ms in duration;weak positive deflection (arrow) of the potential change trace corresponds to a simplenon-overshooting action potential. No luminous activity was observed (upper trace).Fig. 28 B: in the same photocyte, an increase of intracellular depolarizing currentpulse evokes a second response (lower trace) corresponding to the 2-componentovershooting action potential. This response is correlated with a flash (upper trace).

29 30

JIFig. 29. During the intracellular depolarizing current injection (200 ms in duration),repetitive electrical firing (lower trace) and luminous activity (upper trace) wereevoked in a photocyte.Fig. 30. Action potentials induced in the same photocytes as Fig. 29 by one extracellularelectrical pulse applied during 200 ms onto the photogenic area. Note that the luminousintensity of 2 flashes is comparable to that observed during the intracellular stimula-tion of the photocyte.

DISCUSSION

Desmosomes

Desmosomes are classically described in most animal tissues. Belt desmosomes areamong the more widespread intercellular junctions, while spot desmosomes, exten-sively distributed in vertebrate tissues (McNutt & Weinstein, 1973), are absent orpoorly characterized in some invertebrate epithelia such as in insects or coelenterates.

Cell junctions in excitable epithelium 359

31 32

Fig. 31. Demonstration of the propagation of action potentials in the photogenic epi-thelium. The lower trace is the voltage recording of the photocyte into which thecurrent was injected through the double-barrelled microelectrode; owing to raisedfiring threshold (see Fig. 28 B), lower trace amplification was 02 V/div. The uppertrace represents potential changes recorded by a simple microelectrode inserted inanother non-adjacent photocyte. Hyperpolarizing and depolarizing membranepotential changes induced by weak current injections are shown in the 2 epithelialcells. Increase of depolarizing current passage in the stimulated photocytes evokesa response (arrow, lower trace) which is recorded in the neighbouring photocyte(upper trace). This propagated response is a 2-component action potential. Lumi-nescence was not recorded. Microelectrodes were about 120 /tm apart.

Fig. 32. Demonstration of the propagation of action potentials in the non-photogenicepithelium. The upper trace is the current monitor. The middle trace is the membraneresponse of the epithelium cell intracellularly stimulated; note the weak positive de-flection (arrow). The lower trace is the propagated simple non-overshooting actionpotential recorded in another non-adjacent epithelial cell. Microelectrodes were about100 fim apart.

In the scale epithelium, the structure of desmosomes seen in thin section correspondsto that described by Farquhar & Palade (1963) in vertebrate tissues; however, theydiffer with regard to intercellular spacing which, in spot desmosomes, is about 22-35nm in vertebrates (Staehelin, 1974) and can reach o-i /tm in the annelid Harmothoe(Fawcett, 1969).

The most prominent structural characteristics of desmosomes found in the scaleepithelium are 6-7 nm filaments associated with belt desmosomes and 8-10 nuifilaments associated with spot desmosomes. This difference in diameter is similar tothat observed in vertebrate tissues, where the 7-nm filaments were demonstrated to beactin-like (Rodewald, Newman & Karnovsky, 1976) and the 10-nm filaments wereidentified as a-keratin filaments or tonofilaments (Steinert, 1975). In the scale epi-thelium, 8-10-nm filaments are also found in the cell processes on which spot des-mosomes are differentiated. The bundles of 8-10 nm filaments contained in thesecell processes are probably those that appear linked with the cytoplasmic denseplaques of spot desmosomes.

360 A. Bilbaut

Septate junctions

Septate junctions are widely distributed in invertebrate epithelia and were describedinitially by Wood (1959) in Hydra. Subsequent reports have shown their structuralheterogeneity when studied with lanthanum tracer and freeze-fracture techniques.A classification of septate junctions into 3 major types has been proposed by Staehelin(1974) but recently, Green (1978) has reported 5 new structural types, including oneanastomosing type in the endoderm of an echinoderm. The structural types ofinvertebrate septate junctions may be specific to a species such as the Hydra type(Hand & Gobel, 1972; Filshie & Flower, 1977; Wood, 1977); others have beendescribed in several species of the same phylum such as the continuous-type ofarthropods (Noirot & Noirot-Timothe'e, 1967) (the so-called smooth type (Flower &Filshie, 1975)). Some are common to several phyla, such as the pleated type of molluscs(Flower, 1971; Gilula & Satir, 1971) and arthropods (Gilula, Branton & Satir, 1970;Noirot-Timothe'e & Noirot, 1973; Flower & Filshie, 1975).

In the scale epithelium of the worm Acholoe, septate junctions localized betweenepithelial cells show the following structural characteristics: (a) honeycomb-likepatterns in tangential section in conventionally stained preparation; (b) pleated orundulating bands in tangential section after lanthanum impregnation, the periodicityof the undulation being about 23 nm; (c) distinct rows of membrane particles afterfreeze-fracture, the particles being associated with the P face while grooves or pitsoccur on the E face. These different characteristics define the pleated-type septatejunctions in invertebrate epithelia (Staehelin, 1974).

In annelid epithelia, only pleated type septate junctions have been well characterizedwith lanthanum tracer (White & Walther, 1969; Baskin, 1976). According to thework of Welsh & Buchheim (1977), however, other types of septate junctions appearto exist in annelids. These authors have reported a surprising diversity of septatejunctional types in freeze-fractured preparations. Some of the figures showed thepleated-type structure, whereas most of them were not related to any particular type.To characterize this junctional diversity, it would be necessary to use lanthanumtreatment and freeze-fracture techniques in parallel.

In septate junctions, it is accepted that the transverse septa and lanthanum-revealed pleated bands are a single structural entity as seen in cross- or tangentialsection of the junctional area. It is also established that the topographical dispositionof intercellular pleated bands corresponds to that of the rows of intramembranousparticles revealed by freeze-fracture (Noirot-Timothe'e, Smith, Cayer & Noirot, 1978).The most striking features of freeze-fractured septate junctions in the scale epitheliumare the junctional surfaces where disordered rows of membrane particles occur. Suchappearances, revealing the disposition of pleated bands in the intercellular cleft, alsopermit the explanation of the variability of interseptal spacing observed in cross-sections of conventional or lanthanum-treated preparations, as shown in Fig. 10.Septate junctions surround the apical regions of epithelial cells and, as noted withother invertebrate epithelia (Flower, & Filshie, 1975; Noirot-Timothe'e et al. 1978),the pleated bands in the scale epithelium are probably discontinuous structures,

Cell junctions in excitable epithelium 361

since the extremities of some rows of particles are seen in the freeze-fractured septatejunctions.

Examination of replicas has revealed junctional points between 2 rows of membraneparticles which could form anastomosing sites in the septate junctions. However, ifthe topographical disposition of rows of particles is similar to that of pleated bands,at present, there is no evidence proving that the junctional intramembranous particlesare the sites of insertion of these intercellular bands. Consequently, it cannot beconsidered as a certainty that these junctional points correspond to real anastomosingsites in the septate junctions.

Other particular features observed in the freeze-fractured septate junctions in thescale epithelium are the E face membrane depressions lined by a row of membraneparticles and the corresponding P face ridges. Similar figures have been described ininsect epithelia by Noirot-Timothe'e & Noirot (1979); they represent intersectionsbetween 3 cells and are defined by these authors as tricellular junctions.

Gap junctions

In the scale epithelium, gap junctions are extensively distributed between all theepithelial cells. Their structural characteristics revealed by conventional stainingprocedures or lanthanum treatment of the preparation are similar to those generallydescribed in both vertebrate and invertebrate tissues (Staehelin, 1974; Larsen, 1977).However, in these 2 groups, 2 fracturing patterns of gap junctions have been dis-tinguished according to the distribution of the junctional membrane particles on eitherfracture face. In the P type (the so-called A-type), junctional particles remain associatedwith the P face, and in the E-type (the so-called B-type), junctional particles remainassociated with the E face. All the gap junctions described in vertebrate tissue belongto the P-type (McNutt & Weinstein, 1970; Staehelin, 1974; Larsen, 1977), whereasin invertebrate tissues both types are encountered: P-type in molluscs (Flower, 1971;Gilula & Satir, 1971) and E-type in Hydra (Filshie & Flower, 1977; Wood, 1977),chelicerates (Johnson, Herman & Preus, 1973; Lane, 1978a), Crustacea (Peracchia,1973; Hanna, Keeter & Pappas, 1978) and insects (Flower, 1972; Satir & Gilula, 1973;Dallai, 1975). In 2 invertebrate phyla, annelids and platyhelminthes, P-type (Kenstler,Brink & Dewey, 1977; Quick & Johnson, 1977) and E-type gap junctions have beenreported (Flower, 1977; Larsen, 1977).

In this study, junctional particles remained clearly associated with the P face andcomplementary pits with the E face after freeze-fracturing of fixed and glycerinatedepithelium. Consequently, the gap junctions of the scale epithelium are P-type gapjunctions. They can be compared with the P-type gap junctions differentiated betweenglial cell processes of the earthworm (Kenstler et al. 1977) with regard to the distri-bution, arrangement and size of the junctional membrane particles. The diameterof their junctional particles (11-12 nm) is intermediate between that reported in theP-type gap junctions of molluscs (8-5 nm, Gilula & Satir, 1971) and platyhelminthes(12-14 nm, Quick & Johnson, 1977).

Compared with the E-type gap junctions described in annelids (Flower, 1977),similarities are found in the centre-to-centre spacing (about 12 nm) of junctional

24 CEL 41

362 A. Bilbaut

particles and also in their arrangement in a polygonal lattice. Only the fracture faceon which the junctional particles are associated is different. Both gap junction typescould coexist in annelids but curiously in this group and also in platyhelminthes,P-type gap junctions have been revealed after glycerination of pre-fixed tissues(Kenstler et al. igjT, Quick & Johnson, 1977; present study), while E-type gapjunctions have been revealed after glycerination of unfixed tissues (Flower, 1977).This suggests a possible effect of the fixative solution on the fracturing patterns ofgap junctions. Such a possibility can be supported by observations made by Flower(1977) in Hydra and some arthropods showing that treatment prior to freeze-fractureprocedures modifies the distribution of junctional particles on the fracture faces ofthe cell membranes. Furthermore, in a freeze-fractured crustacean epithelium, gapjunctions of the dense type and the loose type have been described (Graf, 1978);in the former, junctional particles are preferentially associated with the P face, whilein the latter, junctional particles are linked rather to the E face. These differentresults seem to indicate that the strict separation of freeze-fractured gap junctionsinto 2 structural types needs to be reconsidered in invertebrate epithelia.

Cell coupling and cell-to-cell action potential propagation

In the present study, electrical coupling between photocytes and between non-photogenic epithelial cells has been demonstrated. Membrane potential changesinduced by weak current injection into one epithelial cell are propagated with decre-ment into neighbouring cells. The low input resistance of the epithelial cell membraneprobably indicates an extensive electrical coupling but because of technical restraintsthe coupling coefficient has not been measured.

All the epithelial cells are electrically excitable; intracellular current injection inphotogenic and non-photogenic epithelial cells elicits action potentials. As a result ofcell coupling, action potentials are propagated in the photogenic area when evokedin photocytes and propagated in non-photogenic epithelium when evoked in commonepithelial cells. Consequently, these observations show that the photogenic area andthe non-photogenic epithelium are conducting and excitable epithelia in which actionpotentials spread from cell-to-cell by way of low-resistance junctions. Conductingand excitable epithelia have also been found in other invertebrates, mainly Cnidaria(Mackie, 1976) and Mollusca (Kater et al. 1978). The present investigation andprevious reports made by Bilbaut (1978a) and Herrera (1977, 1979) confirm theirexistence in Annelida. Further evidence of cell coupling and cell excitability in thephotogenic area of the scales in Acholoe is provided by the amplitude of the luminousresponses, which are comparable whether photocytes are intra- or extracellularlystimulated; furthermore, in the latter case, simultaneous observations of the photo-genic area during a flash have shown that the luminous activity originates in a groupof photocytes (Bassot & Bilbaut, 19776).

In fragmented scales, the intensity of flashes initially stimulated intracellularlyincreases rapidly, then decreases slowly. As previously reported, the increase offlash intensities occurs in the entire extracellularly stimulated scale and results fromthe spreading of an active zone which migrates in the photogenic area, flash by flash.

Cell junctions in excitable epithelium 363

During this period, the propagation of 2-component action potentials could berestricted to the illuminated zone. However, results obtained on the the fragmentedscales seem to indicate that, in all cases, the action potentials accompanying luminousactivity are propagated in the whole of the photogenic area.

The coupling and excitability properties of epithelial cells reported in this studyconfirm previous conclusions resulting from extracellular stimulation of the scaleepithelium (Bilbaut, 1978 a); they are also in agreement with observations made byHerrera (1979) in bioluminescent scales of the polynoid worm Hesperonoe. On theother hand, these results are inconsistent with conclusions drawn by Nicolas, Moreau& Guerrier (1978) in the bioluminescent polynoid worm Harmothoe. These authorsreport that in entire scales, acetylcholine solution added to the bath induces a lightemission which is reversibly abolished only in the presence of antimuscarinic drugs.Luminous inhibition being complete even when the scales are electrically stimulatedwith field electrodes, Nicolas et al. (1978) conclude that the luminescence depends onthe activation of muscarinic receptors and that the photocytes are electrically in-excitable.

Action potential characteristics

Action potentials recorded in the epithelial cells of the scale in Acholoe are similarwhether they are evoked by extra- or intracellular stimulations. Two types of responseoccur. Firstly, simple non-overshooting action potentials are observed in bothphotogenic and non-photogenic epithelial cells; in the latter, the amplitude of responsesis higher than that recorded in photocytes. In the polynoid worm Hesperonoe, Herrera(1979) reports that non-overshooting action potentials have the same amplitude inboth epithelial cell types. Secondly, in Acholoe, 2-component overshooting actionpotentials appear solely in photocytes and at a more positive firing threshold than thatof non-overshooting action potentials. The propagated 2-component action potentialis specifically correlated with luminous activity arising from the photogenic area. InHesperonoe (Herrera, 1978), the waveform of action potentials accompanying theluminous activity depends on whether or not the scale is attached to a metamericsegment. In isolated scales, intracellular stimulation elicits propagated responsesbeginning as one or several spikes. These spikes are followed by a long-lasting depola-rizing plateau which repolarizes abruptly and the duration of this response is between4 and 5 min. If the scale remains connected to the worm, photocyte depolarizationinduces a propagated action potential similar to that observed in isolated scales ofAcholoe; an initial fast spike is followed by a new depolarization. In whole or frag-mented scales in Acholoe, photocyte depolarization has never shown a long-lastingdepolarizing plateau in standard saline solution.

Action potentials of epithelial cells differ not only in their waveform characteristics,but also in their ionic requirements. The non-overshooting action potentials areNa-dependent and TTX-insensitive (Herrera, 1979; Bilbaut, unpublished obser-vations). In Acholoe, the initial overshooting fast spike of the two-component actionpotential is Ca-dependent while the second component is Na-dependent (Bilbaut,19786). Herrera (1979) observed in Hesperonoe that both initial fast spikes and

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364 A. Bilbaut

plateaux recorded in the photocytes of the isolated scales are Ca-dependent. InHesperonoe and Acholoe, the luminous activity is mediated by the entry of Ca2+ ionsinto the photocytes (Herrera, 1977, 1979; Bilbaut, 19786).

Functional implications of intercellular junctions

Most authors agree that desmosomes (belt and spot desmosomes) are involved incell adhesion (review by Staehelin, 1974). In the scale epithelium, the adhesivefunctions of desmosomes appear to play a role at 2 levels. Belt desmosomes, which aredifferentiated between epithelial cell bodies, are probably important for the cohesionof a monolayered epithelium. Spot desmosomes are found in abundance between theepithelial cell bodies and the cell processes containing 8-10-nm filaments. These pro-cesses are comparable to pillars crossing the internal compartment of the scales andsupporting the 2 epithelial layers to which they are anchored by multiple spot des-mosomes. Thus, the structural complex formed by pillars and their spot desmosomesappears to be involved in the cohesion of 2 epithelial planes.

The respective functions of septate and gap junctions in invertebrate epithelia stillremain unclear. As noted earlier, low resistance pathways are involved in electricalcoupling or metabolic cooperation between cells in excitable or inexcitable invertebrateepithelia. These low-resistance pathways probably correspond to membrane speciali-zations found by microscopical observations. Septate and gap junctions always seemto coexist in invertebrate epithelia (Hagopian, 1970; Hudspeth & Revel, 1971;Gilula & Satir, 1971; Rose, 1971) but direct evidence of their functional role islacking. On the contrary, in other tissues, there are good correlations to show thatgap junctions correspond to coupling sites. Vertebrate tissues are considered to bedevoid of septate junctions and their tight junctions are functionally implicated in thetransepithelial permeability barriers (Machen, Erlij & Wooding, 1972; Claude &Goodenough, 1973). Cell coupling has been correlated with the presence of gapjunctions in many cases. These results have been obtained in inexcitable tissues suchas cell cultures (Gilula, Reeves & Steinbach, 1972; Epstein & Gilula, 1977) andexcitable tissues such as cardiac muscle (Barr et al. 1965; Dreifuss, Girardier &Forssman, 1966) or smooth muscle (Barr et al. 1968). In invertebrates, the nervoustissue of crayfish has provided good correlations between structure and function ofgap junctions. Bidirectional (Peracchia, 1973) and rectifying (Hanna et al. 1978)electrical synapses examined after freeze-fracture reveal structural intramembranousorganization comparable to that of the E-type gap junctions. In addition, no excitableelectrically coupled tissue has been shown to be completely devoid of gap junctions,with the possible exception of some smooth muscles (Henderson, Duchon & Daniel,1971).

In Acholoe scales, the presence of gap junctions between cells of a conducting andexcitable epithelium is confirmed. Assuming that in some excitable tissues cell-to-cellpropagation of action potentials is mediated by way of gap junctions, one may con-sider that in the excitable epithelium of the scales these junctions are probably thestructural sites of electrical coupling. Concerning the septate junctions, these haveoccasionally been reported as the only coupling sites in invertebrate epithelia (Bullivant

Cell junctions in excitable epithelium 365

& Loewenstein, 1968; Gilula et al. 1970), but their direct involvement in cell couplinghas not been conclusive. Invertebrate epithelia appear to be devoid of tight junctions- except in the insect rectum (Lane, 19786) - and the apical intercellular cleft opensdirectly to the extracellular medium. At present, certain studies (Lord & Di Bona,1976; Filshie & Flower, 1977; Noirot-Timothee et al. 1978) lead us to believe thatseptate junctions would have a sealing function and would play a role in the controlof ionic transepithelial permeability. Septate junctions in invertebrate epithelia wouldbe physiologically analogous to tight junctions in vertebrate tissues. In the scaleepithelium, if transepithelial ionic movements occur by way of the intercellularspaces, they could flow only between the extracellular medium of the internal com-partment and the external seawater medium. Although the existence of such apermeability has not been demonstrated, the only intercellular epithelial structurescapable of regulating an ionic flow between internal and external compartments of thescales are the septate junctions which surround the apical region of the cells below theextracellular collagenous cuticle.

This work was supported by the C.N.R.S. (L.A. 244 and R.C.P. 431). Freeze-fracturereplicas were prepared at the Laboratoire de Biologie et Technologie des Membranes (U.C.B.);ultrastructural observations were made at the Centre de Microscopie Electronique Appliqu6ea la Biologie et a la Geologie (U.C.B.).

I thank Drs C. Noirot, C. Noirot-Timoth6e and H. Skaer for critical reading of the manu-script. I am grateful to F. Hemming and Dr. C. Wilkinson for correction of the English textand A. Bosch for his assistance in preparing the photographic plates.

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(Received 25 May 1979)