The morphological basis of impulse conduction in a jellyfish epithelium was investigated. Lanthanum impregnation of endodermal canal and endodermal lamella verified the existence of true gap junctions in Polyorchis. In both transverse and en face sections of gap junctions, electron-lucent globules, with a width of 7–8 run and a spacing of about 11 run, are evident. Gap-junctions are concentrated at the peripheral canal margin and septate junctions are localized around the canal lumen. Epithelial cells of the endodermal canals are capable of conducting a non-decrementing action potential. It is suggested that endodermal spike propagation, which can mediate ‘crumpling’ behaviour, is dependent upon low-resistence ionic pathways provided by gap-junctions and upon sealing of the intercellular space from saline extracellular fluids by septate junctions.

Considerable evidence implicates gap-junctions as intercellular pathways mediating electrical coupling in non-excitable epithelia and certain excitable tissues (e.g. crayfish lateral giant axon; vertebrate smooth and cardiac muscle). Gap-junctions in non-excitable tissues probably allow for intercellular transport of metabolites, while in excitable tissue they probably provide low-resistance connexions for the propagation of action potentials. This subject is discussed in reviews by Gilula (1974), Sheridan (1974), and Staehelin (1974).

A number of physiological studies have demonstrated the existence of excitable epithelia in invertebrates (Mackie, 1965; Mackie & Passano, 1968; Kater, Rued & Murphey, 1978), larvae (Roberts & Stirling, 1971; Mackie & Bone, 1976) and chordates (Matthews & Sakamoto, 1975; Mackie & Bone, 1977). There is, however, a scarcity of detailed ultrastructural evidence of specialized intercellular junctions that can be associated with this excitability.

The endoderm of the hydromedusan Polyorchis penicillatus is capable of conducting an action potential (Spencer, 1978). Such epithelial spikes lead to contraction of tentacles, manubrium and gonads together with intorsion of the umbrella margin. This protective behaviour is known as ‘crumpling’ and is commonly seen in many hydromedusae (Mackie & Passano, 1968). This study details the intercellular specializations that are likely to be correlated with this ability of the endoderm to propagate epithelial spikes.

Jellyfish were collected from eel-grass beds in Bamfield Inlet (W. Coast of Vancouver Island) and held in a flow-through aquarium at about 11 °C for a maximum of 5 days.

For conventional fixation, pieces of jellyfish were anaesthetized in a 1:1 mixture of seawater and isotonic (0·33 M) MgCl2 and fixed for 1·5 h at room temperature in a mixture of 2·5 % glutaraldehyde and 2% formaldehyde in 0·1 M cacodylate buffer at pH 7·4 containing 5 mM CaCl2 and 0·23 M NaCl (modified from Karnovsky, 1967). After 3 rinses in 0·1 M cacodylate buffer at pH 7·4 with 5 mM CaCl2 and 0·44 M NaCl, the tissue was dehydrated, postfixed for 2 h at 4 °C in 1 % OsO4 in 0·1 M cacodylate buffer at pH 7·4 with 5 mM CaCl3 and 0·42 M NaCl, rinsed as before, and embedded in Araldite. Thin (silver to grey) sections were stained in 50% ethanol-saturated uranyl acetate for 20 min and in lead citrate for 5 min.

For lanthanum impregnation (Revel & Karnovsky, 1967), lanthanum nitrate was added to the conventional fixative and buffer rinse to give a 2 % solution and this was followed by a 1 h rinse in 0·03 N NaOH (Albertini & Anderson, 1974). The tissue was then postfixed, dehydrated, and embedded as before. Thin sections were either stained in uranyl acetate and lead citrate or examined unstained.

All sections were viewed on uncoated 200-mesh grids in a Philips EM-200 electron microscope.

Two major divisions of the endoderm of Polyorchis can be recognized. Firstly, there is a contiguous canal system consisting of a marginal circular canal with branches into the tentacles, 4 radial canals extending to the bell apex, where branches pass to the gonads, and the tubular endodermis of the manubrium. Secondly, an interradial lamella connecting with the radial canals and with the circular canal is present.

Sections were taken only from the endodermal canals and the endodermal lamella, on the assumption that membrane junctions found here would be representative of the whole endoderm.

Septate junctions

Arrays of septate junctions are prevalent at the lumenal border of the endodermal canals (Fig. 1), and are especially apparent in lanthanum-impregnated material. Although low-magnification micrographs (Fig. 1) suggest a heavy impregnation by lanthanum, higher magnifications (Figs. 2, 3) show that there is either an incomplete penetration or that lanthanum was washed out of these regions during subsequent tissue processing. Lanthanum will, however, thoroughly and permanently penetrate gap-junctions (Figs. 4, 6–10). In transverse section septate junctions show an overall width of 25–26 nm with an intercellular space of 13–14 nm. The septa are approximately 7·5 nm wide and are regularly spaced with periodicities between 14 and 16 nm (Figs. 2, 3).

The septate junctions form numerous belts enclosing narrow cellular processes (Figs. 1–3).

In tangential section, the septate junctions appear as a series of faint (due to poor lanthanum penetration) concentric curves (Fig. 3), demonstrating that they are of the ‘Hydra type’ (Staehelin, 1974).

Septate junctions are also present in the endodermal lamella but no obvious localization within the tissue is detectable.

Gap junctions

In the endodermal canal, gap junctions are normally not closely associated with septate junctions. They tend to be concentrated at or near the periphery of the canals (Fig. 4).

In conventionally stained sections these junctions have a gap of 4–5 nm between apposed outer membrane leaflets and an overall junctional width of 15–17 nm (Fig. 5).

Gap-junctions impregnated with lanthanum are more easily recognized and the ultrastructural details are more evident. It is apparent that lanthanum persists at gap junctions but is washed out from non-junctional intercellular regions (Fig. 6). The central electron-opaque band in these lanthanum-filled junctions is about 7·5–8·5 nm wide and corresponds to the intercellular gap plus the 2 outer membrane leaflets. This is verified by the fact that measurement of this region in conventionally stained gapjunctions gives a width of 7–8 nm. The overall width of lanthanum-penetrated gap junctions (16–18 nm) is comparable to that of conventionally stained junctions. In certain regions of these transversely sectioned junctions, electron-lucent bridges connecting the membranes of adjacent cells can be seen crossing the lanthanum layer (Fig. 6, inset). These lucent bridges have a width of 6–7·5 nM and show a periodicity of about 10–12 nm. At some bridges a central electron-opaque line (ca. 2 nm wide) can be seen passing between membranes (Fig. 6, inset).

In en face sections, gap-junctions appear as plaques containing polygonally packed electron-lucent globules having diameters of 7·5–8·5 nm (Figs. 7–10). The spacing between globules varies from plaque to plaque and within a single plaque. Where globules are widely separated, lanthanum fills the intervening space and polygonal packing is less obvious (compare Figs. 7 and 10 with Figs. 8 and 9). The centre-to-centre distance between globules, when measuring nearest neighbours, is 10·0–11·5 nm. An electron-opaque dot, 2 nm wide, can be seen at the centre of most globules. The star-shaped appearance of many of the globules (Fig. 10) suggests they are composed of subunits, although photographic rotation would be required to determine the precise number (Peracchia, 1973).

Gap-junctions are also common in the endodermal lamella (Fig. 9) but, as is the case for the septate junctions, they show no specific localization within this tissue. The tighter association of septate and gap-junctions within the lamella is probably a consequence of the smaller size (some 4 μm in height as compared to 40 μm for canal cells) of lamellar cells.

Although it has been established that electrical coupling is a general characteristic of epithelia (Gilula, 1974; Sheridan, 1974; Staehelin, 1974), the precise morphological substrate responsible for coupling is often in doubt due to the presence of more than one type of specialized intercellular connexion. In invertebrate epithelia there is a frequent coexistence of septate and gap-junctions (Hudspeth & Revel, 1971; Hand & Gobel, 1972). Nevertheless, it can be inferred from studies on analogous systems, notably the specific action of uncoupling agents at gap-junctions (Peracchia & Dulhunty, 1976; Peracchia, 1977), that gap-junctions are the sites of coupling in epithelia.

Intracellular recordings from the endoderm of Polyorchis (Spencer, 1978) demonstrate its excitability and extracellular records, from a number of other hydromedusae, show that the equivalent epithelial impulse is conducted throughout the endoderm (Mackie & Passano, 1968; Mackie, 1975). Although the coupling ratio has not been measured between endodermal cells in Polyorchis, preliminary recordings (unpublished) have shown that injected current pulses conduct to neighbouring cells. Mackie (1976) has shown that the excitable endoderm of another hydrozoan, Hippopodius, is electrically coupled.

It has been assumed, on the basis of the reported presence of close membrane appositions in conventionally fixed and stained material, that epithelial excitability and propagation are mediated through gap-junctions (Roberts & Stirling, 1971; Mackie & Singla, 1975; Mackie, 1976; Mackie & Bone, 1976, 1977). In Hydra, there is good evidence of epithelial gap-junctions (Hand & Gobel, 1972; Wood, 1977) and indirect evidence of epithelial conduction (Campbell, Josephson, Schwab & Rushforth, 1976). This latter evidence is based on studies of nerve-free (colchicine-treated) Hydra in which no spontaneous activity was recorded although strong mechanical stimulation produced contraction pulses resulting in column shortening. No intracellular recordings have been made to confirm the excitability of epithelial cells in

Hydra

The only studies, that we are aware of, that clearly correlate the presence of specialized junctions with excitability in an epithelium are those of Orci, Unger & Renold (1973) and Matthews & Sakamoto (1975).

The gap-junctions of Polyorchis closely resemble those reported in a variety of invertebrate and vertebrate tissues (reviewed by Gilula, 1974; Staehelin, 1974). It is apparent that the globules seen in en face sections correspond to the electron-lucent bands seen in transverse section, since the widths and periodicities are equivalent. Furthermore, the electron-opaque lines seen passing through the lucent bands in transverse sections (Fig. 6, inset) probably represent the central opaque dot in globules of en face sections. The entrance of lanthanum into these globules is enigmatic in that it suggests incomplete insulation of the intercellular bridge. This does, however, give physical evidence for the existence of a channel which could mediate ionic coupling.

The septate junctions of Polyorchis, in transverse section, are similar to those of other invertebrates (reviewed by Staehelin, 1974). However, in tangential section the smooth, parallel contour of the septa indicates that these junctions are of the ‘Hydra type’ (Staehelin, 1974). This type of septate junction has only been reported in Hydra (Hand & Gobel, 1972; Filshie & Flower, 1977), Pelmatohydra (Danilova, Rokhlenko & Bodryagina, 1969) and Phialidium (Leik & Kelly, 1970); it is apparently restricted to the Cnidaria. In other invertebrate phyla, a second type of septate junction (pleated sheet) is present (reviewed by Gilula, 1974; Staehelin, 1974).

Two functions have generally been attributed to septate junctions, both of which were suggested in the initial description of these junctions by Wood (1959) in Hydra. Firstly, they undoubtedly serve as an adhesion point between cells; secondly, evidence has been provided to suggest that these junctions act as pericellular permeability barriers. As evidence of this latter function, Loewenstein & Kanno (1964) and Josephson & Macklin (1969) obtained electrophysiological data showing the existence of a high resistance barrier between the exterior and lumen of the insect salivary gland and between the exterior and gastrovascular cavity of Hydra, respectively. Septate junctions occur at the lumen of the salivary gland (Wiener, Spiro & Loewenstein, 1964) and at the ectodermal surface and endodermal lumen of Hydra (Wood, 1959; Hand & Gobel, 1972) and this at least suggests their involvement in establishing a transverse permeability barrier. Szollosi & Marcaillou (1977) have correlated the existence of septate junctions in the basal compartment of the locust testicular follicle with the absence of lanthanum and peroxidase penetration into this layer.

In the endodermal canals of Polyorchis, septate junctions encompass numerous cellular interdigitations as described in other hydrozoans (Leik & Kelly, 1970; Hand & Gobel, 1972; Wood, 1959). These interdigitations, by imposing additional complexity on the extracellular space, probably significantly increase the diffusional resistance between the lumen and the mesogloeal face of the canals. Although the septate junctions of Polyorchis are minimally impregnated with lanthanum, they provide little restriction to penetration by the tracer as demonstrated by the thorough impregnation of gap-junctions. Because the endodermal canals are surrounded by mesogloea, lanthanum can reach gap-junctions only via the canal lumen and septate junctions. It is possible that this free permeability is a consequence of fixation. This is supported by the fact that other workers have reported a lack of penetration (Szollosi & Marcaillou, 1977) or minimal penetration (Hand & Gobel, 1972; Filshie & Flower, 1977) through septate junctions when lanthanum is added to living tissue. In addition, Filshie & Flower (1977) have suggested that the septate junctions in Hydra provide a substantial reduction in the permeable intercellular space due to the continuous and extensive nature of the septa in the plane of the cell surface.

In contrast to the canals, the insulating ability of septate junctions in the endodermal lamella is likely to be of lesser importance, since this tissue is presumably insulated from the surrounding seawater by the mesogloea.

One important criterion must be met before an epithelium can propagate an action potential. If the excited state, cell depolarization, is to be relayed to neighbouring cells then sufficient current must flow into the cytoplasm of unexcited cells and across the plasma membrane to the external conducting medium to bring the unexcited cell to spike threshold. The amount of current flowing between cell interiors can be increased by decreasing the resistance formed by intercellular junctions and by increasing the resistance between the cytoplasm and external medium. This latter resistance has 2 major components: the resistance formed by specialized junctional membranes and the remaining non-junctional membrane. We suggest that in Polyorchis endodermal gap-junctions form regions of reduced intercellular resistance and that septate junctions in the canals reduce shunting of local circuit currents by insulating these intercellular ionic bridges from the surrounding seawater. Together these membrane specializations improve the core-conducting properties of this epithelium and allow it to propagate an action potential.

We thank J. McInerney for providing facilities at Bamfield Marine Station and E. Huebner for advice on technical procedure. This study was supported by a National Research Council of Canada grant to A. N. Spencer.

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