Brain Research, 84 (1975) 365-382

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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Research Reports

T R A N S F E R OF RADIOACTIVE M A T E R I A L B E T W E E N E L E C T R I C A L L Y C O U P L E D N E U R O N S OF T H E L E E C H C E N T R A L NERVOUS SYSTEM

E. RIESKE, P. SCHUBERT AND G. W. KREUTZBERG Max Planck Institute for Psychiatry, Kraepelinstr. 2, 8000 Manich 40 (G.F.R.)

(Accepted October 10th, 1974)

SUMMARY

Intracellular application of tritiated precursors by means of microiontophoresis was performed on nerve cells in isolated segmental ganglia of the leech ventral nerve cord. Incorporation as well as intra- and interneuronal transport were studied by autoradiography after injection of fucose, glucosamine, glycine, leucine, orotic acid and uridine. Within several minutes of intraneuronal injection the precursors were incorporated into macromolecules. Depending upon the tracer used, the radioactive material was distributed in a specific pattern over the cell somata and then released into the nerve processes. After application of orotic acid and uridine a transport of radioactive material, presumably RNA, could be observed in the processes of the injected neurons at a distance of about 200-500/zm. Fucose and glucosamine injection resulted in the most extended labeling of the nerve cell processes, indicating a transport rate of about 11 mm/day. When the radiochemicals were injected into one of the two electrically coupled giant nerve cells - - the so-called Retzius cells (Rc) - - a specific labeling not only of the injected Rc but also of the coupled but not injected Rc was found. Injection of protein or glycoprotein precursors into one Rc produced heavy labeling of both Rcs including their processes; a slight labeling of other ganglion compartments was only found after increasing the dosage of the amino acids glycine and leucine. With orotic acid and uridine this interneuronal transfer was confined to the electrically coupled Rc twin. Intracellular injection of one Rc with puromycin followed by injection of amino acids or fucose into the same Rc or into the coupled Rc resulted in an inhibition of precursor incorporation within the puromycin-injected Rc and an exclusive labeling

366 of the coupled Rc, thus indicating that the precursors themselves were transferred. It is suggested that after microiontophoretic application an interneuronal transfer of relatively low molecular weight material takes place, probably across the lowresistance junction through which the Rcs are electrically coupled.

INTRODUCTION

Cells in a number of excitable and non-excitable tissues are coupled by way of low-resistance junctionsS-1°,~l,22,44, 52. In nervous tissue these low-resistance pathways are referred to as electrotonic synapses at which potential changes spread directly from one cell to another with very short delay. Most recent findings indicate that electrotonic synapses between neurons are not confined to invertebrates 19,3°,41,42,51, 67,71 and the lower species of vertebrates2,12-16,21,2s, a4,37 but are also present in the mammalian brain 3,35,65,66. Electron microscopic observations have demonstrated that the morphological correlates of electrical synapses are the 'gap junctions' which appear to contain intercellular channels between the apposed junctional membranes 1,s-l°,aS,~a-56,61. The presence of intercellular channels as the anatomical basis of cellular communication raises the question whether electrotonic synapses of nerve cells permit transmission not only of current-carrying ions but also of larger particles such as organic metabolites. Na, K, Co, CI, I, SOa and sucrose all cross the electrotonic synapse of the crayfish septate axon 11,12,57. Investigations on several kinds of tissues have shown that dyes like Procion yellow, fluorescein, neutral red, Azur B, chromotrope 2 R, Chicago blue, Niagara sky blue and others can pass between cells which are coupled by lowresistance junctions12,22,32,43,44,52,54,5s. However, these tracers have varying degrees of toxicity or bind to cytoplasmic constituents. It seems to be useful to test cell-tocell passage of substances such as metabolic precursors which are of greater physiological interest. From the work of Subak-Sharpe e t al. 69 it is known that genetically deficient B H K 21 cells in culture incorporate tritiated hypoxanthine into their nucleic acid when they are in cytoplasmic contact with BHK 21 cells known to have inosinic pyrophosphorylase activity. The authors suggest that the transferred molecules may have been nucleotide, nucleic acid, the enzyme or substances involved in its synthesis. Kolodny 3a presented some evidence for intercellular transfer of macromolecular RNA between cultured 3T3 cells. Intracellular injection of labeled precursors by means of microiontophoresis combined with subsequent demonstration of the radioactive material by autoradiography has turned out to be a valuable tool for studying incorporation as well as intra- and intercellular transport of material 24,a6,45,46,6a,64. Application of tritiated amino acids by the single cell injection technique to the leech central nervous system most recently showed that radioactive material is transferred not only between glial and nerve cells but also between neurons with electrotonic synapses 2a. Segmental ganglia of the leech are particularly favorable for studying problems concerning intraand intercellular transport. They are not invaded by blood vessels, and have the ad-

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Fig. 1. Semidiagrammatic representation of a transverse section through a segmental leech ganglion with its ventral side at the top (modified slightly after Coggeshall and Fawcett17). Within each of the peripheral compartments the nerve cells (n.c.) and their proximal processes are surrounded by a single large glial cell (g.c.). Nearly all nerve cells send their main process with its fine branches into the dense mass of the central neuropil which contains two large stellate glial cells (s.g.c.), only one of them is represented here. The ventral central compartment contains the two electrically coupled giant nerve cells (Retzius cells, R.c.) which send their processes out of the ipsilateral ganglionicroots. To provide a clear illustration of the course of the Rc processes, their ramifying dendrites are not shown. After an incision is made through the ventral medioanterior part of the neural sheath enveloping the ganglion the glial cytoplasm of this compartment streams out and the two Rcs protrude out of the incision. Histological section (see inset, toluidine blue-eosin, × 403) shows no somatic contact between the Rcs. vantage o f a relatively small n u m b e r o f glial cells a n d n e u r o n s (cf. Fig. 1). M a n y o f the nerve cells can be visually identified a n d f u n c t i o n a l l y characterized 4,5,17,26,27,31, 3s,49-51,59,~s. The largest n e u r o n s a m o n g the a p p r o x i m a t e l y 350 nerve cells i n one g a n g l i o n are the two g i a n t nerve cells, the so-called Retzius cells (Rcs) 6°. They are coupled by a non-rectifying synapse19,30, 40 a n d are t h o u g h t to be neuroeffectors controlling the release o f mucus4L The Rcs c o n t a i n a high c o n c e n t r a t i o n o f 5-hydroxy-

368 tryptamine47,~2 and they lack both electrical and chemical synaptic interactions with Rcs in adjacent ganglia4°. In order to determine if there is a specific material passage between the two coupled Rcs, intracellular applications of various precursor molecules of protein, glycoprotein and RNA were performed on isolated segmental ganglia of two species of leech. MATERIALS AND METHODS

The experiments were performed on fully matured specimens of Hirudo medicinalis and Haemopis marmorata obtained from commercial sources. The leeches were maintained at room temperature. Single ganglia attached to a portion of body wall musculature by their lateral roots were transferred to a Petri dish filled with leech saline6s. The ganglia were pinned to a thin layer of Sylgard 184 resin (Dow Corning) and kept at room temperature. In order to eliminate mutual interaction between Rc and the large glial cell enveloping the two Rcs' somata, the Rcs were exposed by gently making an incision through the ventral central part of the transparent neural sheath surrounding the ganglion. Following this procedure the glial cell of the Rc compartment deteriorated and the Rc somata came into direct contact with the bath fluid (Fig. 1). Control experiments were performed on intact ganglia. Simultaneous recording of electrophysiological activity and intracellular application of precursors were basically the same as described by Schubert et al. 6~,64. The radiochemicals used were [3H]fucose (spec. act. 1.4 Ci/mmole), [aH]glucosamine hydrochloride (2 Ci/mmole), [3H]glycine (2 Ci/mmole), [3H]leucine ( ~ 15 Ci/mmole), [3H]orotic acid (22 Ci/mmole) and [3H]uridine (41 Ci/mmole). Commercial stock solutions (Radiochemical Centre Amersham Buchler) were dried by means of a nitrogen stream and redissolved in acetic acid or potassium hydroxide. In a series of experiments the incorporation of protein precursors was inhibited by pretreating the nerve cells with puromycin. Puromycin (Serva, Feinbiochemica Heidelberg) was dissolved in 0.1 N HCI to give a final concentration of 0.05 M. Iontophoresis current used was in range of 5--40 nA for 3-10 min. After injection the ganglia were kept at room temperature for times ranging from 20 sec to 8 h. They were then fixed by formalin, embedded in paraplast, and autoradiographs were prepared as described by Schubert et al. 63,64. Reconstructions were made by superimposing tracings of the serial radioautographs. TABLE I RESTING AND ACTION POTENTIALS OF RETZ1US CELLS OF TWO LEECH SPECIES

Species

Resting potential* (m V)

Spike height* (m V)

Spontaneously firing Retzius cells (%)

N

Hirudo medieinalis Haemopis marmorata

34 ± 2 41 -~ 1.7

15 ~ 1.5 28 ± 2.2

80 85

119 85

*

.~ (+

S.E.).

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Fig. 2. Radioautograph of a leech ganglion taken 2 h after injection of [3H]glycineinto the central neuropil. Silvergrains which arise mainly from newly synthesizedproteins TM are uniformly distributed over all cells (arrow points towards a stellate glial cell) indicating that the Rcs do not display an exceptionally higher metabolic activity than the other neurons, × 513.

RESULTS

Application o f tracers by means of depolarizing current produces repetitive firing of the Rcs. Hyperpolarizing currents decrease the rates of spontaneous spiking. On average, the resting potentials decrease somewhat immediately after injection. However, several minutes later they reach nearly their initial values indicating that the intracellular injection did not damage the cells. Table I summarizes some electrophysiological parameters of the Rcs. They are electrophysiologically active for many hours even if they are deprived of their surrounding glial cells. The data concerning intracellular incorporation of precursors and intercellular transfer of radioactive material are essentially the same in the two species Hirudo medicinalis and Haemopis marmorata.

Injection of [3H]glycine and [3H]leucine Since specific labeling of Rcs might be achieved due to a putative extraordinary ability to take up precursors rapidly from the extracellular environment, two series of control experiments were performed. In a first series, leucine or glycine were injected into the neuropil of the ganglia. As is shown in Fig. 2, silver grains which arise mainly from newly synthesized proteins is are uniformly distributed over all cells indicating that the Res do not display an exceptionally higher metabolic activity than the other

370

E

371 neurons. In the second series, leucine or glycine were iontophoretically applied with single cell injection procedures to the bathing fluid in the immediate vicinity of the Rc somata, therewith simulating a tracer leakage out of the Rc. No labeling of the cells was detected. Consequently, labeling of a non-injected Rc via the bathing fluid must be excluded under these conditions. When glycine or leucine is injected into one Rc, 20 sec after the end of injection, radioactive material is detectable in its perikaryon and in its main process over a length of about 150/~m. At the same time the coupled but non-injected Rc reveals slight labeling of its perikaryon, whereas its process is labeled only 1-2 min later. In both Rc somata the areas of Nissl substance are labeled preferentially. At this short time interval neither the neighboring nerve cells of the same compartment nor other constituents of the ganglion contain radioactive protein. Fifteen to 120 min after injection of glycine or leucine into one Rc, both Rcs are found to be strongly labeled (Fig. 3A-D). The density of their silver grains exceeds by far that of all other parts of the ganglion. One hundred and twenty minutes after injection, the processes o f the two Rcs can be traced up to 300-400 # m from their somata to the periphery (Fig. 3E). The estimated rates of transport are in the range of 5-6 ram/day. From electrophysiological examinations it is well known that synaptic interaction between the two Rcs is mediated by electrotonic coupling 19,3°,40. However, the morphological correlates of the electrical synapse have not yet been discovered. Yet, we had hoped to find autoradiographic evidence for the existence of supposed areas of contact. As reconstructions show (cf. Fig. 3E), silver grain density is somewhat increased in the region where the Rc main processes enter the central neuropil and then run ipsilaterally to the roots. However, the structural complexity of the neuropil with its intertwined dendrites and spines makes it difficult to relate definitely the radioactive label to the corresponding arborizations of neuronal processes. Transfer from soma to soma of Rc can be excluded, since transneuronal transfer occurs even if the two somata are separated by fine forceps during observation (cf. also Fig. 1, inset, Figs. 4B and 6A). When the amount of the intraneuronally injected amino acid is raised some small neurons of two dorsal compartments contain significantly more radioactive protein than the other neurons (Fig. 3E). To obtain some information concerning the kind of the radioactive material transferred between the Rcs a series of experiments was performed in which puromycin was iontophoretically applied to one Rc. Sixty minutes thereafter amino acid

Fig. 3. Transneuronal transfer of radioactive material 2 h after intraceUular injection of [aH]glycine into one Rc. A-D: autoradiographs of serial transverse sections through a leech ganglion reveal incorporation of amino acid not only in the injected Rc (black arrows) but also in the electrically coupled non-injected Rc (white arrows), × 376. E: schematic reconstruction made by superimposing tracings of serial radioautographs. Radioactive proteins are released down the Rc main process at an estimated transport rate of 5-6 mm/day. Compared to the other nerve cells, small distinctive neurons of two dorsal compartments (small arrows) show increased label indicating a special relationship to the Retzius cells,

372

*t

B

.

.

.

.

.

-°.o

•

Fig. 4. Incorporation o f tritiated uridine and orotic acid within Retzius cells. A : 1 h after intracellular electrophoresis uridine is incorporated into R N A and transported down the process, < 656. B : there is an unequivocal but relatively slight transfer of radioactive material between the two coupled Rcs 1 h after uridine is injected into the left cell. Note the absence o f any somatic contact, /, 650. ( : reconstruction of a ganglion where one Rc (arrow) received orotic acid 2 h before fixation. Radioactive material is strongly confined to the Rc pair.

373 was injected in either the same Rc or in the coupled but untreated Rc. After another 2 h of incubation the ganglia were fixed. Consistent results were obtained with these ganglia insofar that (i) the puromycin-treated Rc revealed complete inhibition of radioactive amino acid incorporation into proteins and (ii) somata and processes of the coupled Rc were heavily labeled. In some instances after previous injection of puromycin into only one Rc, the incorporation of glycine within both Rcs seemed to be nearly completely inhibited. Since the amount of iontophoretically applied puromycin can be calculated only approximately, an injury of the untreated Rc owing to an overdose of puromycin - - and therefore a possible transfer of puromycin - - must be taken into account.

Injection of [3H]orotic acid and [3H]uridine Within 15 min after injection o f orotic acid or uridine into an Rc its nucleus appears densely labeled. Within the next 30 min radioactivity, which in all probability represents newly synthesized RNA, is equally distributed over the perikaryon. One to 4 h after injection the macromolecules appear in the process up to a distance of 200-500 # m (Fig. 4). In comparison to application of protein precursors the Rc processes appear less intensely labeled. Likewise, the transneuronal transfer between the Rcs is comparatively low. Regardless of both the given dosage of R N A precursors and the incubation time, the coupled Rcs contain considerably less label than the injected ones (Fig. 4B and C). In the main, radioactivity is confined to the nucleus of the non-injected Rc. Only a small number o f silver grains are localized over the perikaryon and the proximal part of the process. Nevertheless, the transfer between

Fig. 5. Incorporation of [aH]fucosewithin a Retzius cell 1 h after injection of the tracer. Accumulations of silver grains point to the Golgi complexes where completion of carbohydrate side chains of glycoproteins occurs; this patchy pattern of labeling is absent in the process, × 857.

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Fig. 6. Intra- and interneuronal transport of radioactive material 4 h after injection of ['~H]fucose into one Retzius cell (arrow). A: silver grains which most probably represent newly synthesized fucosyl glycoproteins are mainly confined to the injected Rc (arrow) and the coupled but not injected Rc, × 592. B : section of the same ganglion at the level of the Rc processes. Both processes can be followed up to the initial parts of the lateral roots indicating an estimated transport rate of 11 ram/day. Note the absence of label within the nerve cells of the Rc compartment, 640,

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Fig. 7. Effect of puromycin on incorporation of [aH]fucose and transneuronal transfer. Thirty minutes before application of fucose into the right Rc, puromycin was injected into the left Rc. Two hours after tracer injection the right Rc shows normal incorporation of fucose. From the scarce label associated with the puromycin-treated Rc one can assume that relatively low weight molecular material is transferred from the right Rc to the left one where it is incorporated into some still preexisting polypeptide acceptors for glycoproteins. Puromycin injection 60 min previous to fucose injection results in total absence of label in the puromycin-treated Rc; arrow points towards membrane labeling of the fucose-injected Rc process, × 1008. the Rcs is selective since no other neurons or glial cells reveal labeling after application of R N A precursors into one Rc.

Injection of [ZH]fucose and [3H]glucosamine Fucose and glucosamine differ from all the tested precursors mainly in that they produce the farthest-reaching labeling of the Rc processes. The transported materials are most probably fucosyl glycoproteins. Within a few minutes after administration of fucose to an Rc, radioautographs reveal a characteristic pattern o f distribution of silver grains. Whereas the nucleus is free of label, numerous small accumulations of silver grains are prominent in the perikaryon. These seemingly correspond to the Golgi complexes where glycoproteins are completed6, 29. This patchy pattern of labeling is absent in the process (Fig. 5). Occasionally a heavy label borders the outsides of the nerve cell process (Fig. 7). The labeling pattern of the non-injected Rc is analogous to that of the injected Re (Fig. 6). Thus, both somata (Fig. 6A) together with their processes (Fig. 6B) are heavily labeled. Four hours after injection of fucose the Re processes can be followed

376 up to considerable distances within the lateral roots. Estimates of transport rates are in the range of 11 ram/day. At short time intervals, such as 1 rain after injecting the tracer for 6 min, the estimated values of transport rates were found to lie between 25 and 30 mm/day. Except for the neuropil which contains some diffusely distributed silver grains, no other parts of the ganglion are labeled. When fucose is applied to the glial cell of the Rc compartment, the glial cells of all peripheral compartments contain distinct label. As in the case of amino acids the effect of inhibitors on fucose incorporation and on transneuronal transfer of material was tested. Unfortunately, acetoxycycloheximide, which inhibits the synthesis of polypeptide acceptors for glycoproteins TM, could not be injected by means ofiontophoresis. For this reason puromycin was chosen. It was injected into one Rc either 30 rain or 60 rain before administration of fucose to the coupled Rc. After the tracer injection the ganglia were allowed to incubate for another 2 h. As autoradiographs show, puromycin pretreatment for 30 rain does not completely block fucose incorporation (Fig. 7), whereas puromycin injection 60 min prior to fucose application results in complete suppression of labeling in the Rc. With regard to the labeling pattern of the Rc pair, injection of glucosaminc yields essentially the same results as fucose.

DISCUSSION

The data indicate that after microiontophoresis of all the tested precursors a preferential transfer of radioactive material between electrically coupled neurons takes place. The amino acids are readily incorporated into proteins within the perikaryon, and they are then released into the nerve cell process. For proteins and glycoproteins the estimated transport rates of 5-6 mm/day and 11 ram/day, respectively, are considerably lower than those reported for axonal transport in vertebrate systems (for reviews see refs. 25, 39, 48). A comparably low velocity of 10 mm/day has been described for rapid axonal transport of proteins in the crayfish central nervous systemz0. The silver grains demonstrable after injection of fucose or glucosamine are assumed to represent newly synthesized glycoproteins for three reasons. (i) The relatively high speed of transport corresponds to the values given in the literature for these rapidly transported components 7. (ii) Polypeptide components of fucosyl glycoproteins are synthesized with the participation of ribosomes. They are completed by fucose which is located at the end of some of the carbohydrate side chains. In agreement with the assumption that the completion of these glycoproteins takes place mainly within the Golgi apparatusn, 29 a patchy pattern of labeling is seen in the leech nerve cells after [SH]fucose application. As in the case of cat spinal motoneurons z2 these accumulations of silver grains were thought to represent Golgi complexes. (iii) A distinct increase of label at the surface of the nerve cell somata and processes has been found which agrees with the expectation that some of the glyco-

377 proteins are transported to the plasma membrane to be added to the outer cell coat. Intraneuronal injection of the RNA precursors uridine or orotic acid resulted in heavy labeling not only of the nuclei of the injected nerve cells but also of their processes. The origin of the R N A demonstrable in the processes is not yet clear. From some preliminary experiments using actinomycin D as an inhibitor of DNA-dependent R N A synthesis, we are led to believe that R N A may be synthesized in the soma and then transported down the process rather than being locally synthesized along the process. Our findings provide experimental evidence for distinct passage of molecules between two electrically coupled nerve cells. Depending on the tracer used, the exchange of material takes place to a greater or lesser extent. The question arises whether this transport is strictly confined to the two coupled Rcs or whether glial cells or extracellular pathways are involved. It seems likely that there is a direct passage from neuron to neuron for the following three reasons. (i) For the most part, injections into Rcs were performed after having eliminated the large glial cell of the Rc compartment, thus separating all nerve cell bodies of this compartment from each other. This procedure did not influence the extent of material transfer. In one series of experiments the glial cell of the Rc compartment was not removed. If in this case the glial cell had received some tracer by leakage out of the injected Rc, the glial cell itself should have incorporated at least small amounts of the tracer and should, therefore, have revealed some labeling. However, regardless of the time interval between injection and fixation of the ganglia, the glial cell of the Rc compartment did not show any labeling. The same is true for the two large stellate glial cells of the neuropil. These findings might imply that glial cells do not substantially participate in material transfer between the coupled nerve cell. (ii) Another possible pathway for transfer of ions or molecules could be the numerous extracellular channels penetrating the ganglia. If some tracer were released into the spaces it should be available not only to the Re but also to the neighboring neurons of this compartment which should therefore reveal some labeling, too. This was not found except for the cases of administration of amino acids into the neuropil of the ganglia. (iii) After decoupling of the electrotonic synapse between the Rc pair the transneuronal transfer of radioactive material is specificallyinhibited (unpublished results from Rieske and Hermann). Our conclusion towards a direct transneuronal transfer is supported by the findings that Procion yellow (M.W. about 500) 54 and labeled sucrose (M.W. 342) 11 cross junctional membranes at electrotonic synapses between segments of the crayfish septate axon. Furthermore, the results of these authors indicate that gap junctions which are the morphological correlates of this synapse 'are relatively more permeable for intercytoplasmic passage of larger molecules than the non-junctional membranes are permeable for exchange between cytoplasm and external medium TM. The exact site of coupling between the Rc pair remains to be demonstrated. From light microscopy a contact of cell bodies is unlikely. A somewhat increased amount of radioactive material within the region where the two main processes of the Rcs enter the neuropil points to the possible site of electrical contact in this part of the ganglion. In absence of electron microscopic evidence for the existence

378 of direct contact between the Rc processes, however, the possibility still exists that electrical connection between the Rcs could be mediated by presynaptic fibers of interneurons as is predicted for electrical coupling of neurons of the lateral vestibular nucleus of the rat a~. With regard to the size of the transferred material, it appears from our findings that the precursors themselves or their low molecular weight derivatives are transferred between the two Rcs. This assumption is supported by the data obtained from the short-term experiments and from the experiments in which the synthesis ofmacromolecules was inhibited. The molecular weights of the applied substances are in the range 75 (glycine) to 471 (puromycin). The silver grains detectable in the radioautographs most probably represent newly synthesized macromolecules. Provided that the site of electrotonic coupling is somewhere in the region of densest labeling in the Rc main processes, a supposed transfer of macromolecules in very short-term experiments should result not only in a labeling of the injected Rc soma and its process but also in a labeling of the initial part of the process of the coupled Rc. This was not found. To the contrary, the label first appeared in the soma of the coupled Rc and subsequently in its process. Furthermore, after injection of RNA precursors into one Rc, the coupled but non-injected Rc showed labeling of its nucleus and perikaryon with its process remaining completely unlabeled. These findings are difficult to explain unless it is assumed that low molecular weight material which is transferred via the electrotonic synapse, reaches the soma of the coupled Rc by diffusion and/or retrograde transport and is then incorporated into macromolecules within the cell body. Further evidence for passage of low molecular weight substances is given by the puromycin experiments. If macromolecules were transferred, the puromycintreated Rc should receive at least some label from the coupled Rc to which radioactive precursors of proteins were applied. However, puromycin-treated Rcs never showed any label. The slight labeling of those Rcs to which puromycin had been injected 30 min before application of fucose to the coupled Rc indicates the presence of some still preexisting acceptor proteins for fucose. Puromycin pretreatment for 60 min was sufficient to prevent labeling in this cell. The possibility still remains that molecules of larger size can pass between coupled nerve cells. From the work of Kanno and Loewenstein 3z it is known that substances having molecular weights up to 69,000 (serum albumin conjugated with fluorescein isothionate) can pass across tight junctions between epithelial cells. Nevertheless, there are some difficulties in comparing these findings with our results because of the different kind of tissue and the different injection technique used by these authors. Low-resistance junctions are not confined to excitable tissues but are more prevalent in non-excitable tissues. In an analysis of the possible functional relevance of the low-resistance pathways Furshpan and Potter 2z have suggested that these junctions, in addition to the nearly synchronous spread of electrical signals, have some other basic functions such as distribution of intermediary metabolites and waste products as well as transmission of substances that control movement, rate of division and differentiation. In tissues which lack vascularization, as in the case of the

379 leech central nervous system, the presence of specific pathways allowing rapid exchange not only of ions but also of molecules such as precursors or metabolites appears to be significant. Interestingly, all glial cells of leech ganglia are coupled by lowresistance junctions but do not generate electrical impulses 38. The function of the Rcs is to control mucus release by the skin of the leech 4t. Taking into account that this control of mucus secretion does not necessarily require absolute synchrony of impulse firing, the electrical junction between the Rc pair could have functional relevance for the metabolism of the two Rcs. ACKNOWLEDGEMENT

We express our thanks to Miss Waltraud Komp for skillful assistance and Dr. Roger Eckert for discussion and help in the preparation of the manuscript.

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