The properties and propagation of a cardiac-like impulse in the skin of young tadpoles

Z. vergl. Physiologie 71, 295--310 (1971) �9 by Springer-Verlag 1971

The Properties and Propagation of a Cardiac-like Impulse in the Skin

of Young Tadpoles

ALAN ROBERTS * and CHARLES A. STI~LI~G

Department of Zoology, University of Bristol

l~eceived November 26, 1970

Summary. The contact relationships of skin cells in late embryos and young larvae of Xenopus laevis are described. Superficial cells are joined by ' t ight ' or ' gap' junctions at their outer periphery but elsewhere' simple appositions' arc found. All-or-none impulses are evoked in the skin by electrical or mechanical stimuli (Fig. 3). Evidence is presented in favour of the view that these impulses are generated by the majority of skin cells and not by some neuronal element in the skin. The impulse propagates throughout the skin from any stimulated point (at average speed of 7.7 cm/sec) even when the animal is in distilled water. However, removal of Na+ from solutions bathing the inner skin surface or treatment with Tetrodo- toxin abolishes the impulse indicating that it is Na + dependent. Current injected into skin cells spreads to others so it is suggested that the impulse propagates by direct current flow from cell to cell. The ' neuroid' conduction system in the skin of Xenopus tadpoles is compared to similar systems in coelenterates and to the propagation of the vertebrate cardiac impulse.

I n t r o d u c t i o n

All-or-none impulses p ropaga t ed th rough the skin of young tadpoles (Xenopus laevis, Clawed toad) were descr ibed in a previous note (Roberts , 1969). Three main quest ions abou t the mechanism of the impulse and i ts p ropaga t ion are ra ised by t h a t work. F i r s t ly , where are the impulses genera ted ? Are the membranes of the skin cells exci table , or is some nerve e lement impl ica ted ? Secondly, if the impulses are genera ted b y the skin cells, are t hey p r o p a g a t e d from cell to cell b y direct current flow and if so wha t is the s t ruc tu ra l basis for this ? F ina l ly , are the skin impulses s imilar to o ther impulses in proper t ies such as the ionic currents which underl ie t hem ? I t is wi th these quest ions t h a t this paper is p r inc ipa l ly concerned.

The deve lopment and normal behavioura l funct ion of the impulses will be discussed in deta i l elsewhere (in prepara t ion) . However , we can repor t now t h a t the skin impulses are present before ha tch ing and

* Partly supported by an S. R.C. Fellowship.

296 A. Roberts and C. A. Stirling:

r e m a i n for a t l e a s t t h e f i r s t one or t w o d a y s of l a r v a l life. I n t h i s p e r i o d

t h e i m p u l s e s s p r e a d o u t f r o m a n y s t i m u l a t e d p o i n t , e x c i t e t h e c e n t r a l

n e r v o u s s y s t e m a n d , in t h e h a t c h e d l a r v a , e v o k e s w i m m i n g . T h e s e

o b s e r v a t i o n s i n d i c a t e t h a t t h e e x c i t a b l e s k i n cells a re p a r t of t h e a f f e r e n t

s e n s o r y p a t h w a y of t h e y o u n g t a d p o l e a n d a l low t h e w h o l e su r f ace of

t h e a n i m a l t o be s e n s i t i v e be[ore i t is c o m p l e t e l y i n n e r v a t e d .

Methods

Eggs were obtained by injecting breeding pairs of adult Xenopus laevis with chorionic gonadotrophin and were allowed to develop in aerated tapwater at from 14 to 24 ~ C so t ha t a range of developmental stages were available for a period of two or three days. If necessary the egg membranes were removed using fine forceps. Embryos and larvae were staged using the normal tables of Nieuwkoop and Faber (1956).

For eleetron microscopy embryos were transferred from tap water to buffered fixative, cut or torn into small pieces and placed into fresh buffered aldehyde mixture for two hours. They were washed overnight in buffer and post-fixed in buffered osmium tetroxide for one hour. Fixat ion procedures were carried out at room temperature and solutions were buffered at p i t 7.2 with 0.1 M eacodylic acid. For series (A) fixation was in a 1.6% gluteraldehyde, 1% paraformaldehyde mixture, and postfixation was in 2% osmium tetroxide with 5% sucrose. For series (B) all solutions contained 0.05% CaCI~-6H~O, and D-glucose in place of sucrose. Calcium chloride and D-glucose were used in an a t t empt to improve membrane fixation, bu t no significant change could be a t t r ibuted to them. ttowever, rapid dissection of the tissue into small pieces was crucial for good preservation. Fixed tissue was dehydrated in ethanol, cleared in propylene oxide and embedded in araldite. Sections were cut on an LKB ultramicrotome and stained with uranyl acetate and lead citrate (Fahmy, 1967).

Physiological experiments were carried out at room temperature (18 to 22 ~ C) in g perspex ba th perfused usually with l-Ioltfreter's solution. This has ion concentrations as follows: Na + 62.4 raM, K + 0.67 raM, Ca 2+ 0.9 raM, C1- 64.87 raM, and HCO~ 2.4 mM. In sodium free saline Tris [Tris (hydroxymethyl) aminomcthane chloride] replaced N~C1 and NaI-ICOs. The effect of manganese was studied by replacing 10 mM of NaC1 by MnC12 on an equimolar basis. The pH of these salines was between 7.2 and 7.8.

Recordings were made using conventional techniques with 3M KC1 filled, glass capillary mieroelectrodes. An ~gar saline, silver-silver chloride indifferent electrode was used. Recordings were displayed on a potentiometric pen recorder and also on a storage oscilloscope for photography. Intracellular current injections were measured using a 10 kohm resistor in the ear th line.

Recordings can be made from whole embryos at earlier, non-motile stages bu t after stage 24, two types of preparat ion were used. The first of these, the 'bel ly ' , is made by cutt ing off the head and the dorsal par t including spinal cord, notocord, and myotomes. For embryos and larvae at stages 25 to 38 this gives a preparation consisting of a central mass of undifferentiated yolky tissue with a layer of skin on the uncut surfaces. The 'be l ly ' preparat ion has been used extensively as it is very simple to make. This cannot be said of the second, ' isolated skin ' preparat ion which has only been made reliably from larval stages 33 to 40. The method for making this preparat ion is described in the results.

Impulses in Tadpole Skin 297

Results

1. Anatomy A number of studies have been made recently on aspects of the fine

structure of larval Xenopus skin but none have given details of the contact relationships between the cells (Kelly, 1966; Schroeder, 1970; Steinman, 1968). Since these could throw light on the way in which an impulse propagates from cell to cell we have looked at the structure of the skin, paying particular attention to the cell membranes and their relationships with each other.

The skin of embryos and larvae from stage 22 to stage 40 consists of two layers of cells (see Fig. 1A). Those of the inner or deep layer are flattened and appear to form a fairly homogeneous class. The cells of the outer layer, on the other hand, are more diverse. In trunk regions about 10% of surface cells are ciliated and roughly the same proportion are egg shaped with rather sparse cytoplasm and a very small exposed area at the skin surface. These and the ciliated cells form the most obvious minority categories. The remaining surface cells are roughly cuboidal, typically having fairly dense cytoplasm though there are variations in the general optical density of different cells (see Figs. 1 A and C, and 2B). After stage 30 they have a row of large vesicles lining the outer margin of the cytoplasm at the skin surface (see Fig. 1 B to D). This region stains with alcian blue technique for mucopolysaccharides indicating that the vesicles probably contain mucus.

Details of cell inclusions and the different cell types will not be given as they cannot, at present, be related to the propagation of electrical events through the skin. Further, there seem to be no qualitative differences in the types of intercellular junctions between these different categories of cells. We can therefore describe two general patterns of cell contacts: a) those at the outer surface of the skin, between adjacent superficial cells and forming the terminal bar seen in light microscopic preparations, and b) those found deeper in the skin between superficial cells, superficial cells and deep cells, and between deep cells.

The pat tern of contacts in the terminal bar region is similar to tha t described in most other epithelia (see Farquhar and Palade, 1963). At the outer skin surface, or very close to it, neighbouring cell membranes come very close to each other for a few tenths of a micron. Sometimes the extracelhilar gap is probably occluded (see Fig. 1 B) while in other cases gaps up to 50 ~ or more are left (see Fig. 1 C). Deep to this region of close membranes the intercellular gap widens and there is a series of desmosomes whose characteristics range from those of intermediate junctions, with little intracellular fibrillar material or densification, to complete desmosomes (see Fig. 1 B to D). In addition to the close


Fig. 1A-D


appos i t ion of membranes nex t to the skin surface there are somet imes other areas wi th in the t e rmina l bar where the membranes appear to be in a special close re la t ionship. I n one ease a shor t length of close membrane contac t was found deeper in the skin t h a n the desmosomes (Fig. 1 D). More often the membranes in the t e rmina l ba r run para l le l for shor t s t re tches wi th an i n t e rmembrane separa t ion of 75 to 150 A. Such regions are called " s imp le appos i t i ons" af ter F u r s h p a n and P o t t e r (1968) who suggest t h a t in the absence of a p p a r e n t s t ruc tu ra l special izat ions cellular in te rac t ion is ind ica ted by the fact t h a t the membranes of two cells "run nea t l y in paral lel , even when highly convoluted ".

This t y p e of "simple appos i t i on" is common also deeper in the skin between all types of cells. Some examples are given in Fig. 2. Closer contacts of the " t i g h t " or " g a p " junct ion types have not been found in these places. The appearance of the apposi t ions depends on the general ex ten t of the ext race l lu lar space. When this is ve ry small, as in Fig. 2A, the d is t inc t ion be tween regions of appos i t ion (arrowed) and other regions is unclear . However , when there is more extraee] lular space the apposi t ions s t and out as places where the membranes appea r to be s tuck toge ther (see Fig. 2 B and C). This encourages us in our proposa l t h a t apposi t ions are a site of membrane in terac t ion .

The other t y p e of in tercel lu lar junc t ion found deeper in the skin is the desmosome. Like the apposi t ions , desmosomes have been found inf requent ly be tween all t ypes of cells. An example , wi th an unusua l ly wide cleft, is shown in Fig. 2 B. I t is be tween cells in the deeper layer of the skin.

2. Physiology a) Introduction. Most of the results depend on in t race l lu lar recordings

f rom cells in the skin wi th d iamete rs of abou t 10 ~z. P a r t l y as a resul t of this small size ve ry few cell pene t ra t ions have been stable. Even when t hey were, i t was no t possible to assess the effect t h a t damage had on

Fig. 1A-D. Organization of the skin and contacts in terminal bar region. A. Low power micrograph of belly skin from a stage 25 larva. Intercellular bounderies have been inked over to clarify the cell layers. The outer surface is uppermost and shows the poorly developed vesicular structures at this stage. Dark blobs are pigment or yolk granules. Calibration line 10 it. Series (t~) fixation. B to D. Higher power micrographs of the terminal bar region in a stage 32 larva. Mucus is seen as a filamentous fuzz on the outer skin surface and in larger clear vesicles. Desmosomes are present and some areas of close membrane contact are indicated by arrows. Some "simple apposition" is also present in C. The only example of a close junction somewhat removed from the skin surface is shown in D. Calibration lines 0.2 ~ in B and C; 0.1 it in D. Series (A) fixation. In each picture the space outside the animal

is marked with an asterisk


Fig. 2A-C. Contacts between cells deeper in the skin. A Apposition (arrowed) between cells of a stage 25 larva where extracellalar space is small. Series (A) fixation. B Apposition (arrowed) between a superficial cell and a deep cell with denser cytoplasm. The desmosome is between this cell and another deep cell. Skin of a stage 32 larva. Series (B) fixation. C Appositions (arrowed) in the skin of a stage 25 larva with large extracellular space. Series (B) fixation. Calibration lines

0.2~

resting or spike potentials and possibly also on the interactions of a cell and its neighbours (of. Loewenstein, 1966). Consequently we lack confidence in quant i ta t ive measurements at present, part icularly since experiments were conducted in the arbitrari ly composed I tol t f re ter ' s solution.

b) Recordings /rom Skin Cells. When a spinal larva at stage 33-40 is fastened down in I tol t freter 's solution it is possible, using fine pins, to peel back the skin in a pure condition free of cells f rom the underlying regions. Such flaps of skin tend to curl up but small areas can be held flat using a 0.2 m m diameter stainless steel ring manoevered on a micromanipulator . Inside the ring the inner skin surface is exposed while outside the ring the skin curls over to expose its outer surface. This ' isolated skin ' preparat ion can be stable for m a n y hours.

I n the major i ty of cases, when a mioroelectrode passes through the skin only one negative jump in potential is recorded. This is presumably when the electrode tip is in the outer layer of larger cells. However, occasionally, two negative jumps in potential are recorded as the electrode is advanced, possibly indicating tha t both layers of skin cells have been recorded. Rest ing potentials have ranged up to - -90 mV but more of the


stable penetrations lasting for several minutes have given values between --75 and --85 inV. Similar values are obtained with penetrations from the inside or outside surface of the skin. An example of a recorded penetration is shown in Fig. 3 A (1).

Electrical stimulation of the skin surface with a brief (1 msec) current pulse evokes a characteristic, cardiac-like impulse which can be recorded across the membranes of the skin cells. Some examples are given in Fig. 3A. Once the stimulus strength is sufficient to evoke an impulse, the response is not graded with stimulus strength and the impulse propagates without decrement throughout the sheet of skin. The all-or-nothing characteristic of the impulse is apparent from through- threshold stimulation series [see Figs. 3B(3) and 6C]. They can be evoked by depolarizing extra- or intra-cellular current pulses (e.g. Fig. 4 A). In cells from isolated skin of stage 35-38 larvae, with resting potentials of --75 mV or more, impulse amplitudes ranged from 100 to 114 mV, with overshoots of 12 to 30 InV. They took from 8 to 15 msee to reach their peak and from 42 to 180 msee to decline again to one half of their peak amplitude. The refractory period, to a second like stimulus, was approximately equal to the half decay time of the previous impulse.

The results just presented are from what may be the best penetrations of skin cells. However, many other cells have been recorded and when- ever a negative resting potential is obtained, all-or-none impulses have been present. This applies even to the handful of eases where two negative jumps were seen as the skin was pierced. Though some cells, perhaps for example active ciliated cells, may never have been recorded successfully, it appears that a majori ty of skin cells, large and small, may genera tean impulse.

c) Recordings /tom Attached Skin. Recording conditions in attached skin are less stable than in ' isolated skin' preparations. This may be part of the explanation for the somewhat smaller potentials in attached skin with resting potentials up to --80 mV but more often from --45 to --75 mV and impulses up to 106 mV but more often from 60 to 90 mV. However, the ionic composition of the extracellular compartment of the skin under these conditions is unknown. Further, the slcin is involved in complex transport phenomena one consequence of which is the potential generated across the skin. This is recorded as a variable positive potential when a microelectrode pierces attached skin.

From stage 26 to 38 impulses have been recorded from skin cells over the whole surface of the animal including the eyes, cement gland and fins. Histological examination at the ultrastructural level of fins from larvae at stages between 33 and 38 have shown tha t they are often made up of only three layers, two outer and one inner layer of typical


A 1

0 ! - -

IO0 L _ _ J I min I I 100 m s e c

B1

4

1 . _ _ t I I

mV f ~ - . . . ,

~J - S

L J lOOmsec I I

5

L - - J

3

I I

Fig. 3A and B. Examples of penetration and impulses. A(1) to (g) peeled-off skin. The diagram shows the recording arrangement and A(1) is a pen record of one penetration. Once a resting potential was recorded, an electrical stimulus (1 Inset duration) evoked an impulse. These are not recorded at full amplitude because of the slow response of the recorder. The third impulse (arrowed) is displayed in A (2). A(3) and A(4) show impulses from other ceils in the same skin piece from a stage 35 to 38 larva. A(5) shows an impulse recorded with a 2M K acetate electrode from attached skin of a stage 26/27 embryo. B shows impulses from attached skin. B(1) and B(2) are from a single stage 31 to 34 cell in response to a sharp prod [at arrow in B (1)] and an electrical pulse [B (2)]. B (3) shows a dual penetration of two ceils 1.46 mm apart in a stage 33/34 larva. One stimulus was below threshold for impulse initiation. In each of the oscilloscope records the top trace or line is at zero potential and the stimulus is obvious from the artifact. Calibrations are 20 mV per

division and 100 msecs

skin cells. These therefore appear to be the only cells present from which recordings could have been made in the th inner fin regions. The whole surface of the animal is also sensitive to electrical s t imula t ion and a brief (1 msec) current pulse evokes a single impulse. In some embryos a t the earliest stages when impulses are found (stage 22 or 23), single 1 msec current pulses have sometimes evoked a t ra in of impulses. Sharp prods with a b lun t pin are also effective in evoking impulses [see Fig. 3B(1)] suggesting t ha t nips from predators would be a normal ly effective st imulus.


Conduction velocities for impulses in attached skin have been determined either by simultaneous recording with two microelectrodes [see Fig. 3B (3)] or by sequential recording from a number of cells at different distances from a constant stimulus point. Conduction velocities measured in belly preparations of larvae from stage 31 to 41 ranged from 5 to 11 cms/see with an average velocity of 7.7 era/see.

Impulses have been recorded in bellies of animals from stage 22, when the neural tube has just closed, to stage 41 which is approximately two days after hatching. At the earliest of these stages, resting potentials and impulse amplitudes are smaller. After stage 41 recording becomes difficult and the presence of impulses is uncertain.

d) Propagation o/ the Skin Impulse. By analogy with other systems such as the heart, where impulses are propagated from cell to cell, the most likely mechanism for propagation through the larval skin is by direct current flow from cell to cell. Accordingly, observations have been made to see if this occurs in attached and peeled-off skin.

The procedure was as follows:

1. Two cells were penetrated using separate microelectrodes, and their resting potentials were recorded.

2. One of the mieroelectrodes was then switched from the recording circuit to a pulse generator.

3. Responses to current pulses injected into one cell were then recorded :

a) in the second cell, and b) on withdrawing the microelectrode from the second cell. 4. The current passing microelectrode was switched back to the

record mode to check that it was still intracellular.

In this way spread of current from cell to cell has been recorded in attached skin penetrated from the outside surface and peeled-off skin penetrated from the inside surface. Examples of the records obtained are shown in Fig. 4.

e) Ionic Dependence o/the Skin 1repulse. I t is unusual to find propagated impulses in epithelial cells and it is therefore of some interest to know what similarities there arc between the mechanism of impulse generation in the skin and that in nerve or muscle. Further, it appears that the outer layer of skin cells generate impulses and under normal conditions theh" external surface, roughly one sixth of their total surface, is bathed by pond water. Since conservation of ions would be important to the animal as a whole in this environment, it would be surprising if this outer membrane surface were involved in impulse generation. The question of which cell membranes are responsible for the production of the impulse has therefore been investigated in parallel with its ionic

20 Z. vergl. Physiologie, Bd. 71


f mV i ['x'..~ B ~ _ _ A 0

[ . . . . . . . . . . . ~_____ ~

A0

I J lOOmsec

Fig. 4. Current flow between skin cells in attached (A) and peeled-off (B) skin from stage 35 to 38 larvae. In each record the lowest trace shows the current pulse injected into one cell, the middle trace shows the response inside another cell and the top trace shows the response recorded just outside this second cell after withdrawal of the potential recording electrode. There are therefore two sweeps in each picture but the two current pulses are superimposed. In A an impulse was evoked when the hyper- polarizing current pulse ended. Its peak is indicated by an arrow. The distances

between the current and recording electrodes were (A) 200 B and (B) 80 p

mechanism by comparing the effects of ionic changes and inhibitors on impulses in peeled-off and a t tached skin.

I n the first experiments, te t rodotoxin was used to test the sodium dependency of the impulse. Tetrodotoxin (30 • reversibly abolished the impulse in peeled-off skin where the inner skin surface is exposed (see Fig. 5). The sodium dependency suggested by this experiment was checked by replacing the sodium in the Holtfreter 's solution with Tris. I n one experiment observations were made on a piece of isolated skin. This had a central area where the inner surface was exposed, but outside the holding ring, the skin had curled over on itself to expose only the outer surface. I n this situation, replacement of sodium reversibly abolished the impulse in regions where the inner skin surface was exposed but left the other regions apparent ly unaffected.

The Xenoious skin impulse is in m a n y ways reminiscent of a hear t action potential or impulse. This suggests, among other things, tha t there might be some involvement of eMcinm ions in the impulse like tha t in the frog hear t (Orkand and Niedergerke, 1964). Accordingly, the effect of 10 mM manganese Holtfreter (in which 10 mM of NaC1 were replaced by 10 mM of MnC12) has been tested as it has been shown (Fat t and Ginsburg, 1958) t ha t manganese ions competi t ively inhibit calcium currents. I n isolated skin preparat ions 10 mM manganese produced only a slight change in the skin impulse (see Fig. 6) which was certainly much less than the effect of lower concentrations in other tissues such as frog hear t and barnacle muscle ( tIagiwara and Nakajima, 1966). This suggests

impulses in Tadpole Skin 305

30xlO -gM TTX [ q

vi,- I _J,..--_ 50

, J lOO msec

mins 0 1 3 10 15

Fig. 5. The effect of 30 X 10 -9 )/[ Tetrodotoxin on impulses in peeled-off skin, with the internal surface exposed, from a stage 35 to 38 larva. The records are from different cells and are of single sweeps except for that at 3 rain where a number of

sweeps show the absence of a response as the stimulus intensity was increased

A m y B C

~ !j ' \o~ I' o - - t L__JIO0 rose c t _ _ I lOOmsec ~ - -

D

J t ./

Fig. 6A-D. Examples of impulses in different bathing solutions. A and B are from attached skin on belly preparations where solution changes affect the outside surface of the skin. A is a tracing of an impulse from a stage 25 to 35 larva when Tris replaced sodium, B is from the same stage 26/27 embryo as Fig. 3A(5). I t shows an impulse after 5 rain in distilled water. C and D show impulses from a single cell in isolated skin. C is after l0 rain in 10 mM MnC12 Holtfreter 's and D is 22 rain after return to normal Holtfreter's. The rate of rise of the impulse is faster in D. C has two sweeps, one below and one just above threshold for impulse initiation.

Calibrations 20 mV per division and 100 msec

that the skin impulse in Xenopus is primarily sodium dependent and that the calcium current is much less important than in these other cell types.

The experiment on Tris substitution for sodium, described above, indicates tha t the impulse is only blocked when sodium is removed from solutions bathing the inner skin surface. Other experiments on belly preparations and intact embryos have shown tha t the impulse remains when Tris replaces sodium outside the sldn and also in distilled water when the total ion concentration is very low. Examples of impulses under these two conditions are shown in Fig. 6. Since the skin cells are fairly definitely involved in sodium pumping activities it would be surprising if these changes in external sodium did not produce any changes in the form of the impulse. Itowever, it is clear tha t the inner

20*


membranes of the skin cells can generate a propagated impulse in a variety of external solutions. I t remains unlikely that significant ion currents flow across the complex external surface of the skin cells.

Discussion

Interest in the conduction of impulses through non-nervous epithelia has increased recently since Mackie's (1965) work on the exumbrellar epithelium of hydrozoan medusae. He was able to make extracellular recordings of propagated, all-or-none impulses from this very simple, single layered epithelium where detailed histological s tudy had revealed no indications of nerve cells. So, he concluded that the impulses were generated by, and propagated through, the epithelial cells themselves. In the light of this evidence a number of other "neuro id" conduction systems (Parker, 1919) have been suggested in coelenterates (see review by Mackie, 1971) but in none of these is the evidence so clear cut as in the uniquely simple exumbrellar epithelia. Further, no one has yet managed to make intracellular recordings from coelenterate epithelial cells. Since there is doubt about the identification of "neuro id" conduction systems, at least in these coelenterate cases, it seems worthwhile to review the evidence so far available for 'neuroid ' conduction in the Xenopus tadpole.

Evidence for the origin of the skin impulse in non-nervous epithelial cells :

1. Impulses can be recorded in skin peeled from tadpoles. Such pieces of skin can be cultured for a few days and the impulse remains. Further, ventral ectoderm, showing no response to stimuli, can be removed before the beginning of neurulation and cultured, and after some time in culture the skin becomes excitable and generates impulses.

2. Positive going impulses are only recorded when the potential jumps to a steady negative resting potential. This pat tern of record is typical of the penetration of single cells in many active tissues. I t can be obtained when the skin is penetrated from either surface and at virtually any point on the whole tadpole including the thinnest fin regions where only skin cells are present.

3. Impulses have been recorded in the skin of embryos at stage 22 when histological s tudy by Muntz (1964) has shown tha t the most precocious neurones in the C.N.S. have short processes which could not have reached the skin in the belly region.

This evidence makes an explanation based on any kind of peripheral nerve net seem very improbable. Signs of such a net or plexus have not been seen histologically at stages when impulses propagate throughout the skin. Consequently we feel confident that the impulses are


generated by the skin cells and propagate from cell to cell. Further, the observation that impulses are invariably present when a negative resting potential is recorded suggests that all the cell types in the skin are invaded by impulses, though they may not all actively generate impulses.

The spread of current from cell to cell, which is clear from the current injection experiments, provides a physiological basis for the spread and propagation of the impulse. We must now consider the structural basis for the spread of current. There are two principal possibilities: (a)there could be higher resistance layers at the inner and outer skin surfaces confining currents within the skin and funnelling it from cell to cell or (b) there could be low resistance junctions between the cells. The finding that solution changes at the outside surface of the skin have little effect on the skin cell impulses or resting potentials shows that this outside surface is rather impermeable and has a high resistance like the outer surface of many other eggs and embryos (e.g. Bennett and Trinkaus, 1970). This may be based on the curious vesi- eulated structure of this surface (see Fig. 1B to D and Sehroeder, 1970, Fig. 18) and its mucous coating as well as on special membrane properties. I t must also depend on effective sealing of extracellular pathways to the exterior and this is probably achieved by ' t ight ' junctions near the outer surface (el. Goodenough and Revel, 1970). Such impermeability should be advantageous for an animal living in a dilute medium. On the other hand the inner skin surface seems to have no structural peculiarities. Further, solution changes at this surface rapidly affect skin cell impulses suggesting a negligible diffusion barrier. If only the outer skin surface is of high resistance, current funnelling cannot explain the electrical coupling of cells in peeled-off skin (e.f. Bennett and Trinkans, 1970). We must therefore examine the possibilities for low resistance junctions between skin cells.

Electron microscopy has shown that there are close junctions between cell membranes in the terminal bar region but has not determined whether these are ' t ight ' or 'gap' junctions (Brightman and Reese, 1969). Since these junctions are usually associated with current flow between cells they could be the basis for the spread of current and the impulse. However, we have not found close junctions elsewhere in the skin and this confirms personal communications from Schroeder and Steinman. We have suggested above that all cells in the skin are probably invaded by the impulse yet we have only seen 'simple appositions' and desmosomes between outer and inner layers of the skin cells. Physiological evidence on the properties of 'simple appositions' is not available. Further, ' t ight ' or 'gap ' junctions may have been missed or perhaps separated by the fixation techniques. However it is possible that the situation is similar to that in the Fundulus embryo as described by


Bennett and Trinkaus (1970). The experiments on isolated skin indicate tha t current flows from cell to cell via regions of low intercellular resistance. At present the most likely sites for this are ' t i gh t ' and ' g a p ' junctions near the skin surface and 'simple appositons ' elsewhere. In the intact animal the space inside the animal could form a closed system bounded by the impermeable outer surface of the skin. Such a high resistance layer could therefore lead to some coupling via extracellular pathways which would supplement tha t due to low resistance junctions in a way analogous to that in Nundulus. The resistance of the surface layer, possible leakage pathways and the consequent degree of electrical coupling via this pa thway remain to be examined.

At present our picture of the Xenopus skin conduction system is as follows.

Most cells in the skin of young Xenopus tadpoles are excitable and can generate impulses in response to appropriate stimulation. However the outer surface membrane of the skin is probably inexcitable. The impulse propagates by inducing current flow between skin cells. In innervated cells, the skin impulse excites the sensory neurone and in this way excitation is carried to the CNS.

We see this system as a special adaptation and not as a rationale for the existence of electrical coupling between skin cells.

We have found a similar propagated impulse in young tadpoles of the Common Toad (Bu[o bu]o). The neuroid conduction systems in Xenopus and Bu]o are very much like those described in coelenterates. In both groups an impulse propagates through an epithelial layer to excite the nervous system and evoke a simple response, often an escape response. However in both amphibians the impulse is longer (up to 900 msec in one Bu[o embryo) and the conduction velocity is slower than in the coelenterate cases (average of 7.7 cm/see in Xenopu8 and 4.5 era/see in Bu[o). The slower conduction speeds may result from less specialization in the epithelia of the developing animals. The longer impulse in the tadpoles is probably related to the behavioural response evoked by the impulse. As in the heart, the long duration of the impulse will set a long refractory period for the behavioural responses. In heart and tadpole this avoids a useless flutter and preserves a minimum beat interval essential for effective pumping and swimming.

This possible significance for the shape of the impulse raises the question of the similarity of the skin impulse to the heart impulse. As there is a functional similarity, is there also a common mechanism underlying the impulses ? The evidence available so far suggests that the ionic meehanism for the skin impulse is close to tha t of the frog's heart impulse. There may be slight differences but more evidence on the exact roles of Na + and Ca 2+ is needed before these can be specified. On a more


general level it is interest ing tha t in common with most other excitable tissues the resting potentials of the skin cells are large (round --80 mV). I t could be tha t this results from a decreased P~a/PI~ permeabi l i ty ratio. Will iams (1970) has suggested tha t this ratio is normal ly high in non- excitable cells and theft- resting potent ials correspondingly are low. I t is therefore also interest ing to note t ha t the skin cell resting potent ia l increases at the t ime of neuru la t ion when the skin cells become excitable.

References

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310 A. Roberts and C. A. Stirling: Impulses in Tadpole Skin

Schroeder, T.E. : Neurulation in Xenopus laevis. An analysis and model based upon light and electron microscopy. J. Embryol. exp. Morph. ~3, 427462 (1970).

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Dr. Alan Roberts and Charles A. Stirling Department of Zoology University of Bristol Woodland Road Bristol BS8 lUG, England

The properties and propagation of a cardiac-like impulse in the skin of young tadpoles

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