J. Phygiol. (1977), 273, pp. 691-706 691With 5 text-figure.Printed iniGreat Britain

NON-EQUIVALENCE OF IMPULSE BLOCKADEAND DENERVATION IN THE PRODUCTION OF MEMBRANE

CHANGES IN RAT SKELETAL MUSCLE

BY A. CANGIANO, L. LUTZEMBERGER AND L. NICOTRAFrom the Istituto di Fisiologia, Cattedra II,

University di Pisa, Via S. Zeno, 31, 56100 Pisa, Italy

(Received 18 April 1977)

SUMMARY

1. A complete and long lasting blockade of nerve impulses was estab-lished in the sciatic nerve of rats, by implanting silastic cuffs of criticalinternal diameters. Either marcaine-impregnated or plain cuffs were used.The contralateral sciatic nerve was sectioned.

2. At various days after the initial procedures, the extensor digitorumlongus muscles of the two sides were examined with intracellular electrodes.

3. Decrease in resting membrane potential, fibrillatory activity andresistance of the action potential to tetrodotoxin developed not only inthe denervated but also in the impulse-blocked muscles. In the latter,the fibres were normally innervated since they displayed miniature end-plate potentials and were excitable by nerve stimulation distal to theblocking cuff.

4. However, all of the above mentioned denervation-like changes weresignificantly less pronounced in the blocked muscles than in the denervatedones.

5. It is concluded that in addition to loss of nerve impulses, some otherneural factor must be taken into account to explain the origin of musclechanges induced by denervation. The possible relation of this additionalfactor with nerve degeneration is discussed.

INTRODUCTION

The nature of the neural factors determining the normal properties ofthe skeletal muscle has been the subject of intensive investigation. Suchfactors have been proposed to be either the nerve impulses keeping themuscle active (Jones & Vrbovai, 1970; L0mo & Rosenthal, 1972; Berg &Hall, 1975), or some unidentified neurotrophic substance acting indepen-dently from nerve impulses (Miledi, 1960). Lack of one or the other

692 A. CANGIANO, L. LUTZEMBERGER AND L. NICOTRAmechanism would therefore explain the striking changes that occur in thedenervated muscle fibres, such as appearance of extrajunctional sensitivityto acetylcholine (ACh) (Axelsson & Thesleff, 1959; Miledi, 1960), decreasedsensitivity of the action potential to tetrodotoxin (TTX) (Redfern &Thesleff, 1971 b), decrease in the resting membrane potential (Locke &Solomon, 1967; Albuquerque & Thesleff, 1968) and spontaneous genera-tion of action potentials (fibrillation) (Tower, 1939).

Since chronic stimulation of denervated muscles restores their normalproperties (L0mo & Rosenthal, 1972; Drachman & Witzke, 1972; Purvesand Sakmann, 1974a; L0mo, & Westgaard, 1975; Westgaard, 1975), nodoubt can exist that impulse activity plays a dominant role. On the otherhand, a trophic influence from the nerve is suggested by experiments ofpartial denervation of the frog sartorius muscle (Miledi, 1960) and by theobservation that a longer nerve stump delays the onset of denervationchanges (Luco & Eyzaguirre, 1955; Emmelin & Malin, 1965; Harris &Thesleff, 1972). However, recent results indicating that colchicine appliedto motor nerves induces denervation-like changes in normally innervatedmuscle (Albuquerque, Warnick, Tasse & Sansone, 1972; Hofmann &Thesleff, 1972; Cangiano, 1973) are difficult to interpret, since colchicinecan affect muscle fibres by a systemic direct action (Cangiano, 1973;L0mo, 1974) and without producing a detectable block of axonal transport(Cangiano & Fried, 1974, 1977).To determine whether factors different from impulse activity play a

role in the origin of denervation changes, we have made a quantitativecomparison between the effects of nerve impulse blockade with those ofnerve section. Preliminary accounts of these results have been published(Cangiano, Lutzemberger & Zorub, 1975).

METHODS

Chronic conduction block. A chronic conduction block of nerve impulses wasestablished in male Wistar rats weighing 250-350 g, by implanting silicone cuffsaround the sciatic nerve. Either marcaine-impregnated or plain cuffs were utilized.Plain cuffs were prepared by first thoroughly mixing Dow Corning Catalyst E withthe silicone monomer (Sylastic Medical Grade Elastomer 382, Dow Corning) toobtain a concentration of 0-1 % (w/w), and then placing the fluid silicone in appro-priate moulds where polymerization occurred in about 1 day. Marcaine impregnatedcuffs were similarly obtained, however adding the powdered local anaesthetic(Marcaine, Pierrel) to the fluid silicone at a concentration of 25% (w/w). The cuffshad a length of about 8 mm, outside diameter of 5 mm and inside diameters of1.00, 1.05 or 1-10 mm according to the size of the nerve, cleaned of connective tissue.The cuffs were not sterilized. To avoid trauma of the nerve at the time of appli-cation, the cuff was not only slit longitudinally on one side but was also cut almostcompletely on the opposite side, just leaving a thin bridge which acted as a hinge:the cuff was opened for its placement around the nerve and then gently closed.

MUSCLE DISUSE AND MEMBRANE PROPERTIES 693A plastic sheet was placed around the marcaine cuffs to minimize external diffusionof the drug. The cuffs were kept close by a ligature. After cuff application, frequentobservations were made of the development and persistence of leg paralysis, byexamining the degree of active extension and spread of the digits, the amount ofextensor tone of the foot and the strength of extensor voluntary movements. Withmarcaine cuffs the onset of paralysis was immediate, i.e. it could be observed assoon as the animal recovered from the ether anaesthesia. With plain cuffs the onsetwas delayed: after a period of about 15-20 hr. a paresis of the cuffed leg appearedand became complete in a few hours (that is, approximately 20-30 hr after cuffapplication). The contralateral side was denervated by sectioning the sciatic nerveat the mid-thigh level: this was done at the same time as cuff application whenmarcaine cuffs were used, or when the cuffed leg appeared completely paralysed ifplain cuffs were utilized. With both marcaine and plain cuffs of critical internaldiameters, that is just accepting the nerve (and causing no obvious compression),the paralysis was usually stable throughout the period of examination (up to 15days). If marcaine cuffs of slightly larger internal diameters were used, the paralysiswas short lasting (1-1j days) and followed by a quick recovery to normal functionof the leg. This indicates that it is only initially that the marcaine cuffs work throughan anaesthetic type of block, and that the long lasting impulse conduction block isdue to a mechanism similar to that of the plain cuffs. This mechanism is likely tobe due to a moderate and delayed compression of the nerve (Fowler, Danta &Gilliatt, 1972; Cangiano & Fried, 1977), possibly because the cuffs induce a swellingof the nerve by a mechanical action.

Completeness of the conduction block was tested by recording the isometriccontractions of the extensor digitorum longus (EDL) muscle following sciatic nervestimulation proximal and distal to the cuff. These measurements were performedin vivo, connecting the muscle tendon to a strain gauge and applying single andrepetitive electrical pulses (up to 100/sec) to the nerve. In some animals measure-ments from the contralateral normal side were also obtained, for comparison. In thein vivo acute experiments in which fibrillatory activity and resting membranepotentials were measured, conduction block was also tested for each intracellularlyrecorded muscle fibre (Fig. 3, records to the right) and found present in virtually allfibres (576 over 580 fibres tested in sixteen experiments).In vivo acute experiments. At various days after the above mentioned initial

procedures, the rats were anaesthetized with pentobarbitone sodium and preparedfor in vivo intracellular recordings of fibrillatory activity. Small silastic cuffs con-taining stimulating silver wires were applied around the nerve in order to monitorthe conduction block. One was placed around the sciatic nerve about 1 cm centralto the blocking cuff, the latter being left in siu. A second stimulating electrode wasplaced peripherally to the block, around the common peroneal nerve. The skinwounds were then closed with clips and the wires connected to stimulus isolationunits. The distal third of the anterior surface of the EDL muscle was exposedbilaterally (i.e. on both the cuffed and the denervated side) and the skin flaps wereraised to form a pool which was filled with paraffin oil. Rectal temperature wasmaintained at 38 0C. Temperature of the EDL muscle surface was frequentlymonitored on both sides with a thermocouple and remained between 34-5 and36 0C. Spontaneous and evoked spike activity were recorded intracellularly with3 M-KCI micropipettes, from fibres of the three to four superficialmost layers of themuscle. The frequency of fibrillation potentials in each fibre was determined byaveraging the values measured over several 10 see consecutive intervals. Fibresexhibiting at least one action potential in 10 sec were labelled as spontaneouslyactive.

694 A. CANGIANO, L. LUTZEMBERGER AND L. NICOTRAIn vitro acute experiments. Resistance to TTX of the action potential was deter-

mined in vitro. Both the impulse-blocked and the contralateral deivervated muscleswere placed in a chamber containing a mammalian saline solution at room tempera-ture (23-26 0C), equilibrated with 95% 02 and 5% CO2 (pH 7.1-7.3), which hadthe following composition (mM): NaCl, 135; KCl, 5; CaCl2, 2; MgCl2.6H20, 1;NaHCO3, 15; Na2HPO4, 1; D-glucose, 11. TTX (Sankyo, Tokyo) was added to thebathing fluid to obtain a concentration of 10-1 M. Each fibre was penetrated withtwo micropipettes less than 100 sm apart. One was a 3 M-KCl pipette for recording.The other was a 2 M-K citrate pipette for passing currents. Cathodal pulses wereapplied through the current electrode to depolarize the fibre membrane until thethreshold for spike initiation was exceeded. Before delivering the depolarizingpulse, the fibres were locally polarized, through the same current electrode, at90 mV membrane potential to obtain optimal and constant conditions for thegeneration of the action potential (Redfern & Thesleff, 1971 a). The amount ofresistance to TTX of each fibre was characterized by the maximum rate of rise ofits action potential given by the peak amplitude of the first derivative of the actionpotential obtained with an RC circuit (100 k0, 100 pF). Measurements were madein the impulse-blocked muscles at the end-plate and in the surrounding region(about 1 mm on each side) with similar results. This location also permitted us todistinguish the innervated fibres from those accidentally denervated by the cuff,on the basis of presence or absence of miniature end-plate potentials (min. e.p.p.s).In the denervated muscles, measurements were made in a region corresponding tothat tested in the blocked muscles, that is the region of the original end-plates.

RESULTS

Completeness and stability of conduction blockIn the large majority of rats (85%) which had been implanted with

cuffs of critical size, a behaviourally complete leg paresis developed andremained stable for several days (up to 15 days of examination). This wastrue for both marcaine and plain cuffs, the only difference being a delayedonset in the latter case (see Methods). Completeness of the block wasconfirmed by recording EDL twitch and tetanic contractions followingnerve stimulation proximal and distal to the block. Results from electricalstimulation and clinical observations of paralysis were in excellent agree-ment. Examples of complete (A) and incomplete (B) blockade are shownin Fig. 1. Animals with incomplete paralysis were discarded. Measure-ments of this kind were repeated in a large number of rats at varioustimes after the onset of a behaviourally complete paralysis and the extentof the block measured in each rat. These data are presented in Fig. 2,which indicates that the block was essentially complete throughout thetested period of time, both with marcaine and plain cuffs.

In order to evaluate the suitability of the blocking technique it wasalso important to determine whether and how much nerve damage wasproduced by the cuffs, a minimal amount of denervated muscle fibresbeing desirable. This was done by comparing the magnitude of the EDL

MUSCLE DISUSE AND MEMBRANE PROPERTIES 695tetanic contraction following stimulation distal to the cuff, with that evokedfrom normal contralateral EDL muscles. 'Distal' tetanic contractionswere usually quite vigorous (see Fig. 1A) and their average magnitude

Cuffed sideProximalstimul.

Distalstimul.

Contralateralnormal side

.'I

Single

20/sec

1 00/sec

Single

1 00/sec

[5og

0-1 sec

1 sec

r->_ L.

(..1<

Fig. 1. Completeness of the conduction block induced by cuffs implantedaround the sciatic nerve, tested through EDL muscle tension recordsfollowing nerve stimulation proximal and distal to the cuff. A: completeblockade; plain cuff, 5 days after onset of a clinically total leg paralysis.B: incomplete blockade; plain cuff, 5 days after onset of a clinically partialparalysis. Note, in A, that the 'distal' tetanic contraction of the blockedmuscle is only moderately reduced in respect to normal. The twitch, on thecontrary, is larger (and slower) than normal, a fact attributable to muscledisuse. Accordingly, the trials at 20/sec indicate that the fusion frequencyof the blocked muscle is lower than normal. Note, also, that the twitch ofthe blocked muscle is followed by damped oscillations. This was a usualobservation and might be explained by a synchronous fibrillatory activitydetermined by the nerve-evoked action potential.

A I

B I

[100g

I L

III'

____

696 A. CANGIANO, L. LUTZEMBERGER AND L. NICOTRAin ten rats, examined between 4 and 10 days after onset of the block,amounted to 49-6 % of normal. Considering that some drop in tension isattributable to disuse muscle atrophy (see later), one can conclude thatthe amount of muscle denervation is on the average of moderate degree.Intracellular recordings from EDL muscle fibres confirmed that while insome preparations practically all tested fibres exhibited min. e.p.p.s orwere excitable by nerve stimulation, in others there was a variable per-centage of denervated fibres. Over a total of 225 fibres, impaled in vitroat the end-plate region in seventeen blocked muscles (day 4 to 15), 154(i.e. 68 %) were found to be innervated.

1 000*00O#0 0o * cs0 ..O am oc0o *0 0

0 0 0

0

0

00o 5

0

0 1 2 3 4 5 6 7 8 9 10Days of paralysis

Fig. 2. Stability and completeness of the nerve conduction block inducedby marcaine (filled circles) and plain cuffs (open circles). Measurementsperformed in a series of rats at various days after the onset of a clinicallycomplete leg paralysis. The percentage of block in each rat is obtainedby comparing the magnitude of 'proximal' and 'distal' tetanic contractionsof the EDL muscle (see Fig. 1).

Blocked and denervated muscles showed a comparable amount ofatrophy, confirming previous results (L0mo & Rosenthal, 1972). Intwenty-three paired experiments between day 4 and 14, the averageweight of the EDL muscles was 113-2 + 4-6 mg (blocked; mean + S.E. ofmean) and 116-0 + 4.9 (denervated), as compared to 145-7 + 4*4 for seven-teen normal rats of similar size.

In vivo experiments: Fibrillation and resting membranepotential measurements

Intracellular recordings in vivo from the EDL muscles 5-9 days afterdenervation showed many fibres exhibiting a spontaneous spike activityof the type already described by others (Thesleff, 1963; Belmar & Eyza-guirre, 1966; Purves & Sakmann, 1974b). Much more numerous werehowever the silent fibres. This is of interest because it shows that a

MUSCLE DISUSE.7 AND MEMBRANE PROPERTIES 697

similar situation exists in the in vivo preparation to that described byPurves & Sakmann (1974a) for the rat diaphragm maintained in organculture, in which about one quarter of the fibres were fibrillating. In ourin vivo preparations we found 17-6% of fibrillating fibres in a populationof 609 fibres from sixteen 5-9 day denervated muscles. The great majorityof these fibres (96 %) exhibited a regular pattern of discharge, eithercontinuous or in doublets or triplets, or in bursts. Our general findingsare similar to those of Thesleff & Ward (1975) in the in vivo rat diaphragmand to those of Purves & Sakmann (1974b) in the rat diaphragm in organculture, the only difference being a higher fraction of irregularly dis-charging fibres in the latter preparation.

0 1 sec J40 mV

II|| ill IIl III III II1tII_ ji ;I ~ I I

L1

0-5 sec 5 msec

Fig. 3. Examples of fibrillation potentials recorded intracellularly fromimpulse-blocked EDL fibres which have retained their normal innervation.A and B, records to the left: two fibres of a 6 day-blocked muscle (mar-caine cuff) exhibiting a spontaneous and regular discharge of actionpotentials. Records to the right: each record shows the effect of twosupramaximal electrical shocks applied in rapid sequence to the nerve, oneproximal (first stimulis artifact) and the other distal (second stimulusartifact) to the blocking cuff, during an interval between spontaneousspike discharges. The two fibres are excited only by the distal shock.

Also in the impulse-blocked muscles we found fibrillating fibres, withdischarge patterns entirely similar to those described above for the de-nervated muscles (Fig. 3). Furthermore, the average frequency of dis-charge of the active fibres was comparable in the two situations (7.2 + 0-9spikes/sec in forty-nine blocked fibres, and 7-1 + 0-9 in 112 denervatedfibres, sixteen pairs of muscles). Many of the impulse-blocked fibreswhich were fibrillating were normally innervated (65% of 48 fibres tested

26 PHY 273

698 A. CANGIANO, L. LUTZEMBERGER AND L. NICOTRA

in twelve muscles) in that they showed transmitted action potentialsfollowing sciatic nerve stimulation distal to the cuff (Fig. 3).The similarity of pattern and frequency of the spontaneous discharge

of denervated and blocked fibres, indicates that also in the latter thisdischarge originated from the muscle fibres themselves and not fromthe innervating axons. This was confirmed in a few fibres where intra-venously injected D-tubocurarine (0-1-0-8 mg) abolished the transmittedaction potential evoked by nerve stimulation, without interfering withthe spontaneous discharge of fibrillation potentials.

TALE 1. Percentage of fibrillating fibres in sixteen pairs of blocked anddenervated EDL muscles. M and P: marcaine and plain cuff, respectively

% of fibrillating % of fibrillatingfibres in fibres in

Cuff type Time (days) blocked muscles denervated muscles

M 5 2*6 29*8P 6 1'6 18P 6 6 6M 6 14*9 16*7P 6 8*1 15*5M 7 0 11.5P 7 19*4 30'6P 7 0 13-9M 7 14-6 22*9P 7 3*4 10P 8 32*5 17'5M 8 0 30M 8 2*5 32*6P 9 8.8 5*6P 9 8-3 13'9P 9 0 6.7

Mean +s.E. 7.7±2.2 176±2*3P < 0*005(Student's t test)

As in the denervated muscles, the majority of the fibres impaled inblocked muscles (30-60 per muscle) were not spontaneously active. Whenthe percentage of fibres fibrillating in each muscle was compared betweenblocked and denervated muscles, it was clear however that there were ingeneral fewer fibrillating fibres in the blocked preparations. The resultsfrom sixteen muscle pairs, between 4 and 9 days after denervation oronset of total conduction block, are shown in Table 1. The average per-centage of active fibres was 7-7 + 2-2 in the blocked muscles and 17-6 + 2-2in the denervated muscles (602 and 609 fibres tested respectively), witha high statistical significance of the difference (P < 0.005).

MUSCLE DISUSE AND MEMBRANE PROPERTIES 699

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700 A. CANGIANO, L. LUTZEMBERGER AND L. NICOTRAResting membrane potentials of the fibres in the denervated muscles

were lower than in normal muscles (Table 2), as shown by others in in vitropreparations (Locke & Solomon, 1967; Albuquerque & Thesleff, 1968).Generally, however, resting membrane potentials were higher in our in vivopreparations (see also McComas & Mossawy, 1965) with respect to thoseusually measured in vitro in both normal and denervated fibres. Fibres ofthe blocked muscles also displayed a resting membrane potential lowerthan normal, although significantly higher than that of the fibres in thedenervated muscles. Considering all the fibres of the blocked muscles,irrespective of whether innervated or denervated, one obtains an averagevalue of 69-50 + 0-36 mV to be compared with 67-88 + 0*31 mV for thefibres of the denervated muscles, a small but statistically significantdifference (Table 2). This sets a minimum value for the difference in restingmembrane potential between impulse-blockade and denervation. On theother hand, if one considers only the innervated fibres, that is thoseexcitable by nerve stimulation distal to the block, then the averageresting potential is considerably higher (Table 2), although still lower thannormal. This sets a maximum value for the difference in question. How-ever, this is probably too high a value, since spike initiation can fail insome of the blocked fibres which have the lowest resting membrane poten-tial (McArdle & Albuquerque, 1973). These fibres show only subthresholdend-plate potentials following nerve stimulation, and most of them musthave gone undetected in our in vivo preparations, where we were recordingusually at a considerable distance from the end-plates. Therefore, thetrue value of the difference in resting membrane potential between blockedand denervated muscles, should lie somewhere between the above indi-cated minimum and maximum values (i.e. 1-62 and 6-42 mV, respectively).

In vitro experiments: TTX-reaistant action potentialsAfter denervation, directly elicited muscle action potentials become

partially resistant to TTX (Redfern & Thesleff, 1971b). Chronic impulse-blockade produced in our preparations a similar effect, since the majorityof the tested fibres (85% of eighty-one fibres in ten muscles, day 4-10 ofblock) were found to some degree resistant to the poison. These fibres hadretained their innervation, as shown by the presence of min. e.p.p.s at theend-plate region (Fig. 4). As shown in the preceding section for fibrillationand for resting membrane potential, the blocked muscles again were lessaffected than the denervated ones, that is they were on the average moresensitive to TTX (Fig. 5A). Accordingly, a similar difference in sensitivityto TTX was present in the blocked muscles between the innervated fibresand those accidentally denervated by the cuff (Fig. 5B). In order toinvestigate whether the difference was long lasting or transitory in nature,

MUSCLE DISUSE AND MEMBRANE PROPERTIES 701

in a group of seven rats we compared the resistance to TTX betweenblocked and denervated EDL muscles at 14-15 days. The difference inthis group was much smaller than at the earlier time period and statistic-ally not significant (blocked muscles: 181 + 14-2 V/sec, sixty-four fibres;denervated muscles: 215 + 12-2, ninety-four fibres; P > 0.05).

Finally, a separate group of experiments was designed to observe the

Normal Denervated Blocked

I'I'

i~1JY`ii2_I I40 mV

I 300 V/sec1 e

1I0 msec

A/_I_ WA

I' l fI

V __A' _in

V H

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\-Jo_~~~~~~~~~~~~1 .1

I &

.

0 5 sec

Fig. 4. Examples of the action of TTX on the spike generating mechanismof normal, denervated (5 days) and blocked (5 days, plain cuff) EDLmuscle fibres. The top line is the zero potential level, the middle recordrepresents membrane potential changes, and the bottom record is the firstderivative of the action potential. A: one normal, one denervated and one

blocked fibre in normal saline solution. B: one normal, three denervatedand three blocked fibres under TTX. Note the complete blockade of anyregenerative response in the normal fibre (delayed rectification is observedwith the highest levels of depolarization). Note, in contrast, the decreasedsensitivity to TTX of both the denervated and the blocked fibres. To theright: min. e.p.p.s recorded from the blocked fibres.

A_Cont

Control j"-.) \~vO

B

TTX10-6M

702 A. CANGIANO, L. LUTZEMBERGER AND L. NICOTRAeffects produced on resistance to TTX by the interaction between impulse-blockade and denervation in the same muscle. For this purpose, a groupof six rats was prepared as usual by establishing a conduction block alongthe sciatic nerve of one side with a cuff and by sectioning the contralateralnerve. After 7 days, the paralysed side was also denervated by sectioningthe sciatic nerve just distal to the cuff. After 4 additional days, the resis-tance to TTX was measured in vitro in the EDL muscles denervated sincethe initial operation (11 days of denervation) and compared to that of

40A B

30-

% 20-

10 h-10

0 400 800 0 400 800Max. rate of rise of spikes (V/sec)

Fig. 5. Non equivalence of impulse blockade and denervation. Distributionof resistances to ITX, measured as the maximal rate of rise of the actionpotential, in impulse-blocked and denervated EDL muscle fibres (4-10days). The number of fibres in each class is expressed as percent of thenumber of fibres tested in each population. A, diagonal cross-hatching:81 innervated fibres of ten blocked muscles (four muscles: marcaine cuffs;six muscles: plain -cuffs). A, vertical cross-hatching: 131 fibres of tendenervated muscles. Means: 211 + 18-7 V/sec (mean ± s.E.) for the blockedfibres, and 330 +15-3 for the denervated fibres; P < 0-001. B, diagonalcross-hatching: 52 innervated fibres of seven blocked muscles. B, verticalcross-hatching: 42 denervated fibres found in the same seven blockedmuscles. Means: 250±28 for the blocked, and 377 + 36-7 for the dener-vated fibres: P < 0-005.

the blocked muscles denervated with a delay (7 days of impulse blockadefollowed by 4 days of denervation). The interesting result of this compari-son was that resistance to TTX was significantly higher in the lattergroup (blocked + denervated muscles: 293 + 13-8 V/sec, 101 fibres; dener-vated muscles 231 + 10-6, 104 fibres; P < 0-001). This may suggest that

MUSCLE DISUSE AND MEMBRANE PROPERTIES 703

denervation sets free the action of some factor which summates withinactivity in producing spike resistance to TTX.

DISCUSSION

We have shown here that chronic blockade of nerve impulses is followedby denervation-like changes, such as fibrillation, resistance to TTX ofaction potentials and fall in resting membrane potential in muscle fibreswhich have retained their normal innervation. This confirms and extendsthe findings of other workers showing with various approaches the im-portant role of muscle inactivity in the origin of ACh supersensitivity(Jones & Vrbova, 1970; L0mo & Rosenthal, 1972; Drachman & Witzke,1972; Purves & Sakmann, 1974a; Berg & Hall, 1975), TTX resistantaction potentials (Berg & Hall, 1975), fibrillation (Purves & Sakmann,1974a), and membrane depolarization (Berg & Hall, 1975; L0mo &Westgaard, 1975).Our main finding is however that impulse-blockade, whether induced

by marcaine or plain cuffs, is less effective than nerve section in producingdenervation-like changes, since on the disused side the percentage offibrillating fibres was smaller, action potentials were more sensitive toTTX and resting membrane potential was less decreased than on thedenervated side. Other workers have shown similarly that nerve impulseblockade with tetrodotoxin has a smaller efficacy than denervation inproducing extrajunctional ACh receptors, as measured with a-bungaro-toxin binding (Pestronk, Drachman & Griffin, 1976; Lavoie, Collier &Tenenhouse, 1976; see however Berg & Hall, 1975). The difference ob-served in our experiments might conceivably have been due to an in-complete or not well maintained block. This possibility was however dis-carded by the tests of nerve conduction through the cuffed region, rou-tinely performed (see Methods).The possibility must also be considered that in the blocked muscles

some of the fibres initially denervated by a cuff-induced axonal damage,might have been later reinnervated by the intact axons through collateralsprouting. This situation would conceivably tend to shift the averagevalue of denervation-like changes for the innervated fibres towards thehigher values observed in the denervated fibres. Nevertheless, the averagevalue of the innervated fibres was significantly lower than that of thedenervated fibres of both blocked and denervated muscles.The present findings, taken together with the established role of

muscle inactivity, unequivocally demonstrated with experiments usingdirect electrical stimulation of denervated muscles (L0mo & Rosenthal,1972; Drachman & Witzke, 1972; Purves & Sakmann, 1974a; & L0mo &

704 A. CANGIANO, L. LUTZEMBERGER AND L. NICOTRAWestgaard, 1975; Westgaard, 1975), provide a clear indication that atleast two factors must be involved in the origin of muscle denervationchanges. The difference between blocked and denervated muscles canindeed be explained with the hypothesis that the denervated muscles, inaddition to being inactive, also lack some neurotrophic factor normallyexerting an inhibitory influence on denervation-like changes. Alternatively,one can propose that in the denervated muscle there is some additionalfactor that causes denervation-like changes and summates its effects withthat of inactivity. This factor could be released by the degenerating nerve,as previously suggested (Jones & Vrbova6, 1974). Although a definiteconclusion about these two possibilities awaits further experimentation,our data regarding spike resistance to tetrodotoxin suggest that the secondinterpretation may be correct, since the difference between blocked anddenervated muscles appears to be transitory and delayed denervation ofblocked muscles produces greater changes than denervation alone. Theinterpretation based on products of nerve degeneration is also supportedby recent findings indicating that a transient ACh hypersensitivitydevelops after denervation in directly stimulated soleus muscles (L0mo &Westgaard, 1976) and that partial denervation of the rat EDL muscleproduces denervation changes not only in the denervated fibres but alsoin adjacent innervated ones (Cangiano & Lutzemberger, 1977).

Finally, the question may be asked whether the cuffs also blocked, atleast in part, axoplasmic transport. If this was the case, should one followthe hypothesis of a neurotrophic factor accounting for the differencebetween blocked and denervated muscles, one might conclude that thisdifference would have been greater if the block of transport had not occur-red. Indeed, partial block of transport has been reported to occur at thecuffed region of rat sciatic nerves (Bisby, 1975). However, this finding maywell simply be related to the fact that the cuffs produce degeneration,presumably by compression, of some axons in the nerve. This is indicatedby the presence in some of our blocked preparations of a variable fractionof denervated muscle fibres. Furthermore, it has recently been demon-strated that the threshold dose of colchicine for blocking axonal trans-port, corresponds to that sufficient for inducing axonal degeneration andmuscle denervation (Cangiano & Fried, 1977). It appears therefore un-likely that any significant block of transport occurred in the parent axonsof the many normally innervated muscle fibres found in our preparations.

This investigation was supported by research grants from the Muscular DystrophyAssociation of America and from the Consiglio Nazionale delle Ricerche of Italy.We thank Mr Edoardo Biagetti for invaluable technical assistance.

MUSCLE DISUSE AND MEMBRANE PROPERTIES 705

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