J. Physiol. (1973), 232, pp. 193-208 193With 7 text-figuresPrinted in Great Britain

EFFECTS OF TUBOCURARINE ANDESERINE ON THE AXON-SCHWANN CELL RELATIONSHIP

IN THE SQUID NERVE FIBRE

By JORGE VILLEGASFrom the Centro de Biofisica y Bioquimica,

Institute Venezolano de Investiqaciones Cientificas (IVIC),Apartado 1827, Caracas, Venezuela

(Received 19 February 1973)

SUMMARY

The effects of eserine and D-tubocurarine on the axon and Schwanncell membrane potentials have been studied in the giant nerve fibre ofthe squid.

1. The addition of eserine at concentrations of up to 10-4M to theexternal sea-water medium has no appreciable effects on either theSchwann cell electrical potential of unstimulated nerve fibres or on theresting and action potentials of the axon.

2. However, eserine at a concentration of 10-9 M prolongs the long-lasting Schwann cell hyperpolarizations which follow the conduction ofimpulse trains by the axon.

3. Higher concentrations of eserine (10-7, 10-4M) decrease and blockthe long-lasting effects of nerve impulse train conduction.

4. D-tubocurarine at concentrations of up to 10-5 M has no appreciableeffect on the resting and action potentials of the axon.

5. However, D-tubocurarine at a concentration of 10-9 M blocks com-pletely the hyperpolarizing effects of nerve impulse trains on the Schwanncell electrical potential.

6. In addition to its blocking action, D-tubocurarine induces transienthyperpolarizations in the Schwann cells of unstimulated nerve fibres bothin intact fibres and in slitted preparations.

7. These findings suggest that a cholinergic system, which may belocated at the axon-Schwann cell boundary, is involved in the genesis ofthe long-lasting Schwann cell hyperpolarization caused by the conductionof nerve impulse trains by the axon.

7-2

JORGE VILLEGAS

INTRODUCTION

It has been shown that the conduction of nerve impulse trains by theaxon is followed by a prolonged hyperpolarization of the Schwann cellsin the giant nerve fibre of the squid (Villegas, 1972). Although the increasein potassium concentration in the axon-Schwann cell space during thenerve impulse trains cannot account for these potential changes, the exist-ing evidences obtained in voltage clamped axons suggest that currentoutflow from the depolarized axon may be responsible for initiating thelong-lasting delayed hyperpolarizations of the Schwann cells in these nervefibres (Villegas, 1972).The presence of some form of coupling between Schwann cells and the

axon has been suggested to account for the mechanism responsible forthe Schwann cell hyperpolarization. Such possibility appears to befavoured by the presence of certain membrane specialized regions whichmight serve as intercellular junctions between Schwann cells and theaxon. Thickenings of the axolemma and narrowings of the axon-Schwanncell space were observed in nerve fibres of different squid species (Villegas& Villegas, 1968). The local thickenings of the axolemma resemble thepost-synaptic densities which have been assumed to be the point oftransmission in synapses where a chemical mechanism is responsible forintercellular coupling. It is unknown if these structural specializations aredirectly related to the axon-Schwann cell interactions described above.However, it appears worth while to explore the possible existence ofchemical coupling mechanisms between the axon and the Schwann cell.The presence of cholinesterases and the effects of compounds acting on

the acetylcholine system have been extensively studied in squid axons(for references, see Nachmansohn, 1959; Ehrenpreis, 1964; Rosenberg,1971), specially after Nachmansohn proposed the acetylcholine theory ofbioelectricity to account for the mechanism responsible for axonal con-duction. Although the intervention of acetylcholine in the conductingmechanism has been ruled out (Ritchie & Armett, 1963), the questionremains of what role, if any, cholinesterases play in the functioning of thenerve fibre.

In the present experiments we have determined the effects of eserine(physostigmine) and D-tubocurarine on the axon and Schwann cell mem-brane potentials in the giant nerve fibre of the squid, Sepioteuthis sepioidea.The evidence presented here shows that at a concentration of 10-9 M inthe external sea-water medium D-tubocurarine blocks the long-lastingeffect of impulse train conduction by the axon on the Schwann cell mem-brane potential, whereas at the same concentration eserine prolongs thelong-lasting Schwann cell hyperpolarization. However, higher concentra-

194

AXON-SCHWVANN CELL RELATIONSHIP 195

tions of eserine also decrease and block the hyperpolarizing effect of axonimpulse trains on the Schwann cells.

METHODSGeneral procedure

Giant nerve fibres with a diameter of 280-400 ,um obtained from the hindmoststellar nerve of the tropical squid, S. sepioidea, were used in all experiments.

Immediately after decapitation of the living squid the nerve fibre was dissectedout of the mantle, placed in a Lucite holder containing artificial sea water, andfreed from all adherent nerve fibres. The giant nerve fibre was kept under slighttension by means of threads tied to both ends. In those experiments involving theisolation of the nerve fibre sheaths, sharp microscissors were inserted into the axonand the nerve fibre was cut lengthwise over several millimetres. All the experimentswere carried out at room temperature (20-22° C).

Experimental solutions(a) Artificial sea water (cf. Hodgkin & Katz, 1949) was used as normal medium.

The concentration of its components in mm was as follows: NaCl, 441; KCl, 10;CaCl2, 11; MgCl2, 53; NaHCO3, 2-5.

(b) Sea-water solution containing D-tubocurarine was prepared by adding thenecessary amount of Intocostrin (E. R. Squibb & Sons, New York) to the media.

(c) Sea-water solution containing eserine was similarly prepared. Eserine sulphate(Sigma Chemical Co., St Louis, Mo.) was used.

Measurement of the electrical potential differencesThe effects of axon impulse trains on the Schwann cell membrane potential were

explored as described in a previous work (Villegas, 1972). The Schwmann cell mem-brane potential was measured before and after prolonged axon stimulation. Theaxon membrane potential was continuously monitored.The experimental procedure utilized to measure the electrical potential differences

across the Schwann cell membrane and across the axon membrane was similar tothat described and discussed in previous works (Villegas, Villegas, Gimenez &Villegas, 1963; Villegas, 1972).

Glass micropipettes were filled with 3 M potassium chloride solution by boilingunder reduced pressure. Connexion was made via an Ag-AgCl electrode. The criteriafor acceptable micropipettes were suitable shape, tip resistance between 12 and40 MQ, and tip potential between 0 and 5 mV.The exploring micropipette was connected to an oscilloscope (Tektronix 565) and

a strip chart recorder (Yokogawa 347), via a neutralized input capacity compensatedamplifier (Type NF 1, Bioelectric Instruments, N.Y., 1011 Q input resistance). Anearthed reference electrode, Ag-AgCl in 3 M potassium chloride solution, connectedto the bath through a sea-water agar-bridge was used. The exploring micro-electroderesistance was measured at frequent intervals during the experiment. The axoncould be stimulated at will using two external fine platinum wires connected viaa stimulus isolation unit (Type ISA 100, Bioelectric Instrument, N.Y.) to a pulsegenerator (Tektronix 161).

JORGE VILLEGAS

RESULTS

Effect of eserine on the electrical potentialsEserine was used to investigate whether cholinesterases are directly

involved in the mechanism responsible for the long-lasting delayed hyper-polarizations observed in the Schwann cells around stimulated axons.Eserine is known to inhibit cholinesterase activity specifically (Nachman-sohn, 1959). However, at concentrations higher than those needed to in-hibit the esterases, eserine appears to also antagonize the action of acetyl-choline (Dettbarn & Davis, 1963). At concentrations below 10-3 M eserinehas no apparent effect on the axon membrane potential in the squid nervefibre (Rosenberg, 1971).The Schwann cell and axon electrical potentials were measured in

S. sepioidea nerve fibres immersed first in normal sea water and then insea-water solutions containing eserine at concentrations of 104, 10-7 or10-9 M. The electrical potentials of several of the Schwann cells surround-ing a single giant axon were successively measured by brief impalementsfrom inside the axon before and after the conduction of nerve impulsetrains. The axon membrane potential was continuously monitored. Eserinewas added to the external medium 10 min prior to the stimulation of theaxon.The addition of eserine to the external sea-water medium has no

appreciable effect on the Schwann cell electrical potential in the unstimu-lated nerve fibres. The membrane potential of 186 Schwann cells of sixteenresting nerve fibres was obtained during the first 10 min after the additionof the drug. Eserine concentrations of 104, 10-7 and 10-9 M were used inthese experiments. No changes of the Schwann cell potential and of theresting and action potentials of the axon, during the -whole experimentalperiod, could be found at these eserine concentrations. However, as willbe described below, the long-lasting effects of nerve impulse train con-duction on the Schwann cell membrane potential were appreciably modi-fied in the presence of the drug.

Fig. 1 shows the results of four different experiments in which the effectsof nerve impulse trains on the Schwann cell membrane potential in thepresence of different concentrations of eserine in the external mediumwere determined. The graphs show that 10-9 M eserine appears to prolongthe effects of the nerve impulse trains on the Schwann cell electrical poten-tial, whereas 10-7 M eserine decreases both the amplitude and the durationof the Schwann cell potential changes, and 104M eserine completelyabolishes the long-lasting effects of axon stimulation.The results obtained in three different groups of five nerve fibres each,

196

AXON-SCHWANN CELL RELATIONSHIP

Control

Trains

I I I --I

10-m eserine

_ I . i I 1_0 10

10'm eserine

104M eserine

._ I I --I I

20 0 10 20

Time (min)Fig. 1. Effect of eserine on the hyperpolarization of the Schwann cell follow-ing conduction of nerve impulse trains by the axon. The electrical poten-tials have been plotted as a function of time. Each graph corresponds tothe results obtained in a different nerve fibre. The axon membrane potential(continuous line) was monitored. Each point corresponds to the potentialdifference recorded in a different Schwann cell. At zero time eserine was

added to the external sea water medium. During the interval indicatedby the vertical bar stimuli were delivered to the axon at 125/sec. Thehyperpolarization observed in 10-9 M eserine is longer than that in the con-

trol nerve fibre. Higher concentrations of eserine decrease (10-7 M) or

abolish (10 4M) the hyperpolarization.

197

-20

-60

E

C

C

0

'a

UV*_

-20

-AO

-60

-40 .* 000000SO 0 IDN"'s% f0

treated as described above, are given in Fig. 2. The Schwann cell hyper-polarizations observed in these nerve fibres following the conduction ofimpulse trains by the axon appear to be prolonged in duration in thefibres immersed in 10-9 M eserine-sea water as compared to those in thecontrol group, whereas both the amplitude and the duration of theSchwann cell hyperpolarization are decreased in the fibres immersed in1O-7 M eserine-sea water. In a group of seven nerve fibres similarly exposedto the action of I04 M eserine, the long-lasting effects of nerve impulsetrains on the Schwann cell membrane potential were completely abolished.

12 Control 10-'M eserinp 10-7M eserineSi . ~~~~~~~(42)(52)

.0 (4ij3) (4|(2)8

i .6~ l |(37) - 11| 11(31)

84 - 1| * ~ * * * !I -^(25)e (41) * * * I * I

0~ -1

0-2 24 4-6 6-8 0-2 2-4 46 6-8 0-2 2-4 4-6 6-8Time interval after axon stimulation (min)

Fig. 2. Effect of eserine on the hyperpolarization of the Schwann cellfollowing the conduction of nerve impulse trains by the axon. The valuesare the means±sx,. of mean of the potential changes recorded in theSchwairm cells impaled (number of cells in parentheses) during each 2 miinterval. Each graph presents the results obtained in a different group offive nerve fibres stimulated as described in Fig. 1.

The results obtained with 10-9 M eserine appear to indicate thatcholinesterase activity may be directly involved in the mechanism re-sponsible for the long-lasting delayed hyperpolarizations observed in theSchwann cells around stimulated axons. The experiments with concentra-tions equal or greater than 10-7 M eserine further suggest that acetylcho-line may also be involved in the genesis of the hyperpolarizationphenomenon.

198 JORGE VILLEGAS

AXON-SCHIWANN CELL RELATIONSHIP

Effect of curare on the electrical potentialsTo investigate whether the effects of eserine described above may

actually be ascribed to an interference of this drug with cholinergicmechanisms present in these nerve fibres, D-tubocurarine was used.Curare alkaloids compete with acetylcholine for specific receptors in thecell membrane (Jenkinson, 1960). However, a large dose of curare alsohas slight inhibitory action on cholinesterase (Changeux, 1966). At con-centrations below 10-2 M curare has no appreciable effect on the axonmembrane potential in the squid intact nerve fibre (Rosenberg, 1971).

In the present experiments, the Schwann cell and axon electricalpotentials were measured in S. sepioidea nerve fibres immersed first innormal sea water and then in sea-water solutions containing D-tubo-curarine at concentrations of 1 0-7, 10-9, 10-10 or 10-12 M. As for the pre-vious experiments, the electrical potentials of several of the Schwann cellssurrounding a single giant axon were successively measured by brief im-palements from inside the axon before and after the conduction of nerveimpulse trains. The axon membrane potential was continuously monitored.Tubocurarine was added to the external sea-water medium 10 min priorto the stimulation of the axon.

Fig. 3 shows the results of one experiment on the effect of 10-9 M-D-tubocurarine. This figure shows that the Schwann cells become hyper-polarized within 5 min after the addition of the drug, and that theygradually return to the initial levels within the next 10 min. It also showsthat once the Schwann cells have recovered from their transient hyper-polarizations, the conduction of nerve impulse trains by the axon has noappreciable after-effect on the Schwann cell membrane potential. Theresting and action potentials of the axon remained unchanged during thewhole experimental period.

Fig. 4 shows the results obtained in a group of five nerve fibres exposedto the action of 10-9 M-D-tubocurarine. The graphs represent the frequencydistribution of the measurements of individual cells obtained in the nervefibres at rest during the initial 10 min immersion period in normal seawater (control) and during the successive 2 min intervals after the additionof the drug to the external medium. The Schwann cell electrical potentialsin these groups of fibres also return to the initial levels within 15 min afterthe addition of the drug.

Fig. 5 shows the results of five different experiments in which the after-effects of nerve impulse trains on the Schwann cell membrane potentialin the presence of different concentrations of D-tubocurarine were deter-mined. The graphs show that D-tubocurarine induces a transient hyper-polarization in the Schwann cells in the nerve fibres at rest, and blocks the

199

200 JORGE VILLEGASlong-lasting effects of nerve impulse trains on the Schwann cell membranepotential. The effects of D-tubocurarine on the Schwann cell membranepotential appear to be a function of the concentration of the drug, beingmaximal at concentrations above 10-9 M, and unnoticeable at 10-12 M.

-20Trains Axon

D-tubocurarine (10-'M) * Schwann

=-4O r eI

-60 _ ______| _______

.a

0 10 20 30 40

Time (min)

Fig. 3. Effect of D-tubocuranine on the Schwann cell membrane potentialof the resting nerve fibre and on the hyperpolarization of the Schwann cellfollowing conduction of nerve impulse trains by the axon. The axon mem-brane potential (continuous line) was monitored. At the time indicated bythe arrow, D-tubocurarrne was added to the bathing fluid at a concentra-tion of 1O-9 M. Each point corresponds to the potential difference recordedin a different Schwann cell in the same nerve fibre. During the intervalindicated by the vertical bar the axon was stimulated as described inFig. 1. After the addition of the drug the Schwann cell became hyper-polarized for several minutes and then returned gradually towards itsinitial potential level. The hyperpolarizing effect of nerve excitation wasabolished by the incubation in 10-9 M-D-tubocurarine.

Even at the highest concentration used in these experiments (104 Mw),D-tubocurarine had no appreciable effect on the resting and action poten-tials of the axon.

These results appear to indicate that curare-sensitive receptors aredirectly involved in the mechanism responsible for the long-lasting delayedhyperpolarizations recorded in the Schwann cells around stimulatedaxons.

AXON-SCHWANN CELL RELATIONSHIP

60

40 -

20 -

ControlM=-40-0 mVS.D. =0 6 mVn=56

0 1

6-8 minM=-447 mVS.D. =4-0 mVn=27

_ 0-2 min ~ ~8-10 min

40 - M=-40 9 mV _ M=-41-9 mVcS.D.=22mV S.D. =3-3mV

W 20 j 1 nn=222011100

2-4 min 10-12 min_ M=-44-7 mV IM=-40-9m\401- S.D. =33-3 mV S.D.]= 1 2 mV

tL_ ~~~n-A41 n=1I8

20

0

4-6 min

40 - M=-47-2 mVS.D. =38 mV

_ n=37

20 -_-40 -50 -40

12-14 minM=-39-5 mVS.D. =10 mVn=15

I-50

Schwann cell membrane potential (mV)

Fig. 4. Frequency distribution of the electrical potential measurements ofindividual Schwann cells in resting nerve fibres, before (control) and duringsuccessive 2 min intervals after the addition of D-tubocurarine to thebathing fluid at a concentration of 10-9 M. M = arithmetic mean; S.D. =standard deviation of an observation, and n = number of observations.Results obtained in five nerve fibres at rest. After the addition of the drugthe Schwann cell became hyperpolarized for several minutes and thenreturned gradually towards its initial potential level.

201

In-HeI 0- 1a

JORGE VILLEGAS

Effect of D-tubocurarine on the Schwann cell electrical potential in slittednerve fibresTo investigate whether the initial hyperpolarization induced by

D-tubocurarine in the Schwann cells in the unstimulated nerve fibresrepresents a direct action of the drug on the cell membrane, the effectsof 10-9 M-D-tubocurarine on the Schwann cell electrical potential in slittednerve fibres were determined.The results of a typical experiment in which the Schwann cell mem-

brane potential was recorded during brief impalements directly from theaxonal face of the sheaths before and after the addition of D-tubocurarineto the external sea-water medium, are given in Fig. 6. After the additionof the drug the impalements gave Schwann cell membrane potentialshigher (hyperpolarization) than those recorded during the immersionperiod in normal sea water, gradually decreasing to the initial levels.Similar results were obtained in four additional nerve fibres.

Fig. 7 is a representation of the frequency distribution of the electricalpotentials of individual cells recorded during three successive 15 minintervals in this group of five slitted nerve fibres. The diagram at the topcorresponds to the measurements obtained in the slitted nerve fibresimmersed in normal sea water. The diagram at the middle corresponds tothe measurements obtained during the first 15 min interval after the addi-tion of 10-9 M-D-tubocurarine to the external sea-water medium. Thediagram at the bottom corresponds to the second 15 min interval afterthe addition of the drug.From these experiments it seems possible to conclude that the transient

hyperpolarizations induced by D-tubocurarine in the Schwann cells in theintact nerve fibres at rest, represent a direct action of this drug on the

Legend for Figure 5.

Fig. 5. Effect of D-tubocurarine (D-TC) on the Schwann cell membranepotential of the resting nerve fibre and on the hyperpolarization of theSchwann cell following conduction of nerve impulse trains by the axon.Each graph corresponds to the results obtained in a different nerve fibre.Each point corresponds to the potential difference recorded in a differentSchwann cell. At the time indicated by the arrow, D-tubocurarine wasadded to the bathing fluid. During the interval indicated by the verticalbar the axons were stimulated as described in Fig. 1. At a concentrationof 10-12 M-D-tubocurarine has no apparent effect on the Schwann cellmembrane potential. Higher concentrations of the drug induce a transienthyperpolarization of the Schwann cells in the resting nerve fibres anddecrease (10-10 M) or abolish (10-9, 1O-7 M) the hyperpolarizing effect ofaxon stimulation.

202

AXON-SCHWANN CELL RELATIONSHIP 203

Trains-40 uControl (ASW) 0

D-TC (10-7M)

-40

-50 [

E(10-'m)

Ira

4I . I_.1...I _I

4.O040 *e0 20s

Eu

V -50FE

C ~~~~~~(10'1M)

W-40 o0o0.* * ge e 0.%

-50F

(lO'1M)

I-40 9 *ee.00 Avns.%P

-50

0 10 20 30

Time (min)

Fig. 5. For legend see opposite page.

204 JORGE VILLEGAS

Schwann cell membrane. These results appear to indicate the existence ofcurare-sensitive receptors in the Schwann cell membrane.

DISCUSSION

The present results are complementary to previous work (Villegas,1972) on axon-Schwann cell interaction in the squid nerve fibre. Theresults show that the long-lasting hyperpolarizing effects of the conductionof impulse trains by the axon on the Schwann cell membrane potential

-20

E

.C D-tubocurarine (IO-9M)o I4~~ ~ ~ ~~*S

E * O

0i

40 00 @00

C

0 10 20 30 40Time (min)

Fig. 6. Effect of D-tubocurarine on the electrical potential of the Schwanncell in nerve fibre sheaths obtained by cutting the axon lengthwise. Theslitted axon was immersed in artificial sea water and at the time indicatedby the arrow, D-tubocurarine was added to the bathing fluid at a concen-tration of 10-9 M. Each point corresponds to the potential differencerecorded in a different Schwann cell impaled from the axonal face of thesheaths. After the addition of D-tubocurarine the Schwann cell becamehyperpolarized for several minutes and then returned gradually towards itsinitial potential level.

are appreciably modified by the external application of compounds actingon cholinergic systems. At a concentration of 10-9 M in the external sea-water medium D-tubocurarine blocks the long-lasting effects of axon im-pulse train conduction, whereas at the same concentration eserine pro-longs the long-lasting Schwann cell hyperpolarizations. However, higherconcentrations (10-7, 104 M) of eserine also decrease and block the hyper-polarizing effects of axon impulse trains on the Schwann cells. Thus,cholinergic mechanisms appear to be directly involved in the genesis of

205AXON-SCHWANN CELL RELATIONSHIP

60M=-39-8 mVS.D. =0-9 mVn=56

40 _

20 _-

0

60 r-Ca

r_0(U

-o00

OA

(U0

ba

40F-

M=-45 3 mYS.D. =49 mVn=76

kit._-

20 F

0

M=-40-8 mVS.D. =2-1 mVn=43

40

20

0-35 -45 -55

Schwann cell membrane potential (mV)

Fig. 7. Frequency distribution of the electrical potential measurements ofindividual Schwann cells in nerve fibre sheaths obtained by cutting theaxon lengthwise. Top graph: nerve fibre sheaths immersed in normalartificial sea water. Middle graph: 0-15 min interval after the addition ofD-tubocurarine to the bathing fluid at a concentration of 10-9 M. Bottomgraph: 15-30 min interval after the addition of the drug. M = arithmeticmean; S.D. = standard deviation of an observation, and n = number ofobservations. Results obtained in five nerve fibres.

60 r

JORGE VILLEGASthe long-lasting delayed hyperpolarizations observed in the Schwann cellsafter the conduction of nerve impulse trains by the axon.

It was also found that the addition of D-tubocurarine to the externalmedium is followed by a transient hyperpolarization of the Schwann cellsboth in intact nerve fibres and in nerve fibre sheaths obtained by cuttingthe giant axon lengthwise. Thus D-tubocurarine appears to have a directaction on the Schwann cell membrane. At present we have no criticalevidence to account for the underlying mechanism of this transienthyperpolarization. However, this effect of D-tubocurarine on the Schwanncell electrical potential and the abolition of the long-lasting effects of axonstimulation show the presence of curare-sensitive receptors in the Schwanncell membrane. The high sensitivity of these receptors to D-tubocurarinestrongly suggests that they are similar to acetylcholine receptors. Furtherexperimental evidence is needed to settle this point.The presence of acetylcholinesterase (AChE) in the stellar nerve of the

squid has been demonstrated both by chemical and histochemical methods(Nachmansohn, 1959; Brzin, Dettbarn, Rosenberg & Nachmansohn, 1965)in the nerve fibres of other squid species. The results of histochemicaland microanalytical studies still in course in our laboratories (G. M. Ville-gas, J. Villegas and G. Camejo, unpublished) confirm the presence ofAChE in the cell membranes of S. sepioidea nerve fibres. The results of thecytochemical localization by electron microscopy of the regions ofacetylcholinesterase activity show that the dense copper thiocholine endproducts appear mainly distributed along the axolemma forming densepatches attached to the inner leaflet of the membrane. This distributionpattern of the acetylcholinesterase activity resembles those found for thelocal thickenings of the axolemma in intact S. sepioidea nerve fibres andfor the cytochemical localization of regions of adenosinetriphosphatase(ATPase) activity (Sabatini, DiPolo & Villegas, 1968) in Doryteuthis pleinerve fibres. The presence of eserine at a concentration of 10-9 M in theincubation medium resulted in an almost complete absence of acetyl-cholinesterase activity dense end products. These unpublished experi-mental findings appear to give further support to the evidence presentedabove on the apparent involvement of the acetylcholine system in the axon-Schwann cell interactions previously described.

In a recent report, Dennis (1972) describes the effects of depolarizingpulses applied to the Schwann cells on frog muscle denervated end-plates,and interprets them as being due to acetylcholine released from thesuddenly depolarized Schwann cell. Whether the axon and Schwann cellsin the normal intact squid nerve fibre contain, and are able to release acetyl-choline as a result ofnerve impulse train conduction is unknown at present.However, the presence of acetylcholine-like receptors in the Schwann cell

206

AXON-SCHWANN CELL RELATIONSHIPmembrane indicated by the highly specific action of D-tubocurarinedescribed above on the Schwann cell membrane potential, appears tofavour such a possibility.

If the long-lasting delayed hyperpolarizations of the Schwann cellsaround stimulated axons are indeed due to the release of a chemicaltransmitter, then the nature of the transmitter and ionic mechanismsunderlying such an effect remain to be determined.

It is clear that the interactions between Schwann cells and the axonare not simple. Without an understanding of the mechanism, the func-tional significance of the Schwann cell hyperpolarization cannot beassessed. Nevertheless, the experiments herein described have shown thatcholinergic factors are directly involved in the genesis of the long-lastingeffects of axon impulse train conduction on the Schwann cell membranepotential, which represents a further advance in the study of the relation-ship between the axon and Schwann cells that surround it.

I wish to thank Drs Raimundo Villegas, Gloria M. Villegas, German Camejo,and Flor V. Barnola for many helpful suggestions and discussions at different stagesof the work. My thanks are also due to Drs Carlo Caputo and Guillermo Whittem-bury for reading and criticizing the manuscript. I also appreciate the technicalassistance of Mrs Belkiss Pizani, Mr Nehmias Mujica and Miss Elisa Rodriguez.

REFERENCES

BRZIN, M., DETTBARN, W. D., ROSENBERG, P. & NACHMANSOHN, D. (1965). Cholin-esterase activity per unit surface area of conducting membranes. J. cell Biol. 26,353-364.

CHANGEUX, J. P. (1966). Responses of acetylcholinesterase from Torpedo marmoratato salts and curarizing drugs. Molec. Pharmacol. 2, 369-392.

DENNIS, M. (1972). Electrically evoked release of acetylcholine from Schwann cellsat denervated motor end-plates. J. Physiol. 226, 79P.

DETTBARN, W. D. & DAVIS, F. A. (1963). Effects of acetylcholine on axonal con-duction of lobster nerve. Biochim. biophys. Acta 66, 397-405.

EHRENPREIS, S. (1964). Acetylcholine and nerve activity. Nature, Lond. 201, 887-893.

HODGKIN, A. L. & KATZ, B. (1949). The effect of sodium ions on the electricalactivity of the giant axon of the squid. J. Physiol. 109, 37-77.

JENKiNSON, D. M. (1960). The antagonism between tubocurarine and substanceswhich depolarize the motor end-plate. J. Physiol. 152, 309-324.

NAcHMANsOHN, D. (1959). Chemical and Molecular Basis of Nerve Activity. NewYork: Academic Press Inc.

RITCHIE, J. M. & ARMETT, C. J. (1963). On the role of actylcholine in conduction inmammalian nonmyelinated nerve fibers. J. Pharmac. exp. ther. 139, 201-207.

ROSENBERG, P. (1971). The use of snake venoms as pharmacological tools in studyingnerve activity. In Neuropoisons: Their Patophysiological Actions, ed. SIMPSON, L.,vol. 1. New York: Plenum Press.

SABATINI, M. T., DIPOLO, R. & VILLEGAS, R. (1968). Adenosine triphosphataseactivity in the membranes of the squid nerve fiber. J. cell Biol. 38, 176-183.

207

208 JORGE VILLEGASVILLEGAS, J. (1972). Axon-Schwann cell interaction in the squid nerve fibre.

J. Physiol. 225, 275-296.VILLEGAS, G. M. & VILLEGAS, R. (1968). Ultrastructural studies of the squid nerve

fibers. J. yen. Physiol. 51, 44-60s.VILLEGAS, R., VILLEGAS, L., GImENEz, M. & VILLEGAS, G. M. (1963). Schwann celland axon electrical potential differences. Squid nerve structure and excitablemembrane location. J. gen. Physiol. 46, 1047-1064.