extrajunctional ACh sensitivity and TTX resistance when combined with chronic

Transcription

1 J. Physiol. (1984), 355, pp With 8 text-ftgure Printed in Great Britain INTERACTION OF INACTIVITY AND NERVE BREAKDOWN PRODUCTS IN THE ORIGIN OF ACUTE DENERVATION CHANGES IN RAT SKELETAL MUSCLE BY A. CANGIANO, P. C. MAGHERINI, E. PASINO, M. PELLEGRINO AND R. RISALITI From the I8tituto di Fisiologia, Universita di Pisa, Via S. Zeno, 31, Pisa, Italy (Received 27 March 1984) SUMMARY 1. The action of nerve breakdown products on innervated fibres of soleus and extensor digitorum longus muscles was investigated with the following procedures: (1) partial denervation, (2) sensory or sympathetic denervation, (3) section of a previously transplanted foreign nerve. Each procedure was performed either in isolation or combined with chronic muscle inactivity obtained by blocking impulse conduction along the sciatic nerve. Silastic cuffs containing tetrodotoxin (TTX) and sodium chloride were utilized for the block. 2. Partial denervation induced extrajunctional sensitivity to acetylcholine (ACh) and resistance to tetrodotoxin not only in the denervated but also in the innervated fibres. The effects in the innervated fibres were equal in magnitude to those in the denervated fibres, provided they were paralysed. The onset of the membrane changes was synchronous in the two classes of fibres and their amount correlated with the extent of partial denervation. If the innervated fibres were normally active, the membrane changes were still detectable, but considerably smaller than in the denervated fibres. 3. Sensory denervation (removal of dorsal root ganglia L4 and L5) was followed by the development of moderate ACh supersensitivity and TTX resistance in chronically paralysed muscles. Furthermore, section of radicular nerves (total denervation, i.e. efferent plus afferent) induced larger membrane changes than those observed following section of ventral roots alone (efferent denervation). 4. Sympathetic denervation was ineffective even when associated with chronic muscle paralysis. 5. Section of a previously transplanted mixed nerve (superficial fibular) was ineffective if the soleus muscle was normally active, while it induced marked extrajunctional ACh sensitivity and TTX resistance when combined with chronic paralysis of the muscle. 6. Section of a transplanted sensory nerve (sural) also induced extrajunctional membrane changes in paralysed soleus muscles, but their magnitude was much smaller than after section of mixed nerves. 7. We conclude that products of nerve destruction, especially those ofmotor axons,

2 346 A. CANGIANO AND OTHERS induce membrane changes of striking magnitude when potentiated by muscle inactivity. Such an action may also explain the greater efficacy of denervation Vs. pure inactivity, at least at early times after their onset. INTRODUCTION Denervation induces marked changes in skeletal muscle and the nature of the involved neural signals has long been the subject of a lively debate. As far as the extrajunctional membrane changes are concerned (supersensitivity to acetylcholine and related membrane changes: Axelsson & Thesleff, 1959; Miledi, 1960; for a review, see Purves, 1976), lack of impulse or contractile activity certainly plays an important role since chronic direct stimulation can normalize the altered membrane properties of denervated muscles (L0mo & Rosenthal, 1972; L0mo & Westgaard, 1976; for a review, see Fambrough, 1979). On the other hand, inactivity induced in muscle by blocking nerve conduction is less effective than denervation in altering the membrane properties (Cangiano, Lutzemberger & Zorub, 1975; Lavoie, Collier & Tenenhouse, 1976; Pestronk, Drachman & Griffin, 1976; Cangiano, Lutzemberger & Nicotra, 1977; Gilliatt, Westgaard & Williams, 1978). It has been suggested that lack of nerve-borne chemical factors contributes to the origin of denervation changes: possibilities range from materials carried by axonal transport (Albuquerque, Warnick, Tasse & Sansone, 1972; Hofmann & Thesleff, 1972, see however: Cangiano, 1973; L0mo, 1974; Cangiano & Fried, 1977; for a review, see Fambrough, 1979) to the neurotransmitter acetylcholine (ACh), per se (Mathers & Thesleff, 1978; Bray, Forrest & Hubbard, 1982; Drachman, Stanley, Pestronk, Griffin & Price, 1982). Alternatively, several studies have suggested that nerve breakdown products are the factor that accounts, together with inactivity, for the full-blown effect of denervation on the extrajunctional muscle membrane (Vrbovai, 1967; Jones & Vrbova, 1974; L0mo & Westgaard, 1976; Cangiano & Lutzemberger, 1977; Brown, Holland & Ironton, 1978). In the present paper we report the results of experiments conducted on innervated muscle fibres exposed to degenerating nerve tissue coming from different sources: the muscle's own nerve supply by means of partial denervation, sensory nerves and foreign nerves transplanted on the muscle which do not make synaptic contact. Extrajunctional membrane changes (development of ACh receptors and of action potentials resistant to tetrodotoxin, TTX) of striking magnitude always occurred in these circumstances provided the muscle was paralysed by means of a conduction block of its innervation. A preliminary account of some of these experiments has been presented (Cangiano & Lutzemberger, 1980). METHODS All experiments were carried out on adult male Wistar rats. Usually the experimental muscle was the soleus, but some of the experiments were repeated in the extensor digitorum longus muscle (e.d.l.). The action of nerve degeneration products on innervated muscle fibres was tested in various ways: (1) partial denervation, (2) sensory or sympathetic denervation, and (3) section of a previously transplanted foreign nerve. In some preparations innervated muscle fibres were not

3 DENER VATION-INDUCED CHANGES IN MUSCLE 347 interfered with, and were presumably normally active; in other preparations all muscle fibres were rendered inactive by a chronic conduction block along the sciatic nerve. Surgical procedures Ether anaesthesia was used throughout. Partial denervation. L4 and L5 are the two major roots of the sciatic nerve. In order to partially denervate the soleus muscle, the radicular nerve L5 was cut on one side, just outside the vertebral canal. After h, % of fibres in nine soleus muscles (thirty to fifty fibres recorded per muscle) were found denervated as judged from the absence of miniature end-plate potentials (m.e.p.p.s) at the end-plate region. To examine the effect of partial denervation on the e.d.l. muscle, the radicular nerve IA, instead of L5, was sectioned. In a specific series of experiments (Fig. 2) the effect of varying the degree ofpartial denervation over a large range, 7-94 %, was tested. The lowest degrees of partial denervation were obtained by incompletely sectioning the radicular nerve L5, whereas the highest were achieved by cutting completely L5 and incompletely L4. In another series of experiments, a drastic shortening of the latency between partial denervation and membrane changes in the soleus muscle was desirable. This was obtained by introducing, in the routine preparation of partial denervation plus paralysis, two modifications. First, the conduction block of sciatic nerve was established 36 h before partial denervation, which thus acted on a pre-paralysed, highly reactive, soleus muscle. Secondly, partial denervation was obtained by incompletely sectioning the soleus nerve, instead of cutting the radicular nerve L5; it is in fact known that the shorter the peripheral nerve stump, the earlier the onset of degeneration of the distal nerve (Miledi & Slater, 1969) and of muscle membrane changes (Harris & Thesleff, 1972; Uchitel & Robbins, 1978). Sensory denervation. Two approaches were followed. A straightforward one consisted in the ablation of dorsal root ganglia L4 and L5. To avoid damage to the adjacent ventral roots, in later operations only the dorso-medial half of each ganglion was removed. Access was gained through a small laminectomy. The second approach was, conceptually, less direct, but eliminated the problem of accidental damage to the motor axons: we compared the effects of cutting only the ventral roots (efferent denervation) with those ofsectioning the radicularnerves ('total 'denervation, i.e. efferent plus afferent). The two operations were done on different sides of the same animal. The length of the peripheral nerve stump was the same. Sympathetic denervation. The peritoneal cavity was opened through a mid-line incision of the abdominal wall; the most caudal thoracic sympathetic ganglion and the lumbar sympathetic chain were removed, bilaterally. Transplantation and section offoreign nerves. Foreign nerves were transplanted onto the surface of the soleus muscle in young rats (50-80 g body wt.) to allow ample time for recovery from local muscle alterations. The foreign nerve was slipped into a space opened, through a lateral approach, in the fascia overlying the soleus muscle near its proximal tendon. Direct trauma to the muscle was avoided. Foreign nerves used were superficial fibular and sural as examples of motor and sensory nerve, respectively. Section of foreign nerves was made after 1-2 months, the rats having grown to a much larger size ( g). Degeneration of the fibular axons was induced by cutting the common peroneal nerve in the popliteal fossa. For the sural nerve two approaches were used: in one rat the nerve itself was cut; in the other rats the ipsilateral dorsal root ganglia L4 and L5 were removed instead, to avoid degeneration of the few motor axons (maximum five) contained in the sural (Betz, Caldwell & Ribchester, 1980). In the case of the fibular nerve, no synapses were expected to form between the foreign motor axons and the soleus muscle after transplantation, because the muscle remained innervated by its own nerve. Trauma at the same time of transplantation, however, could conceivably induce local membrane changes resulting in hyperinnervation of some muscle fibres by the foreign motor axons. This possibility seems unlikely because: (1) in control experiments, no fibres excitable by foreign nerve stimulation were detected by intracellular recording, and (2) a histochemical stain for acetylcholinesterase (method of Buckley & Heaton, 1968), performed in each experimental muscle after the electrophysiological experiment, did not reveal any staining in the region of the transplanted nerve, but only in that of the original one. In one group of experiments we tested the action of an anti-inflammatory agent, dexamethasone Na phosphate (Decadron, Merck S.D.), on the muscle response to degeneration of the transplanted nerve. The drug was injected in vivo in the lumbar muscles twice a day, 2-4 mg kg-i day-i, a dose corresponding to the highest therapeutic amount in humans. The treatment started at the time

4 348 A. CANGIANO AND OTHERS of foreign nerve section and continued for about 3 days, i.e. up to the electrophysiological experiment. Chronic conduction block Conduction block was obtained by slipping around the sciatic nerve a silicone rubber cuff impregnated with tetrodotoxin (TTX). The cuffs utilized represent a modification of those used by Lavoie, Collier & Tenenhouse (1977). A powder resulting from lyophilization of a watery solution of TTX and NaCl is mixed with the fluid silicone (Medical Grade Elastomer 382, Dow Corning) blended with the appropriate catalyst (M) at a concentration of 0-2 % (w/w). The mixture is placed in cylindrical Plexiglas moulds whose walls have been wet with the catalyst and left to polymerize overnight. The use of NaCl does much more than just helping to handle the minute quantities of TTX. In fact, the cuff in situ progressively absorbs water from the environment, transforming the compact rubber, through which the TTX could not diffuse, into a fine sponge. Excessive NaCI, however, makes the release of TTX too rapid: a 10 % concentration of NaCl in the rubber (w/w), was optimum. The implanted cuffs enlarge, more than doubling their initial size (1 1 mm i.d., 3 0 mm o.d., 4 mm length) in a few days; the increase in i.d. is essential to avoid compression and axonal degeneration resulting from nerve swelling (Cangiano et al. 1977; Cangiano, L0mo, Lutzemberger & Sveen, 1980). Thus the cuff, containing a longitudinal slit, was placed around the nerve and no ligatures were used to keep it in place. Cuffs made with the above procedure can contain a maximum of about 5,tg TTX (0-02 % w/w) without intoxicating the animal; the conduction block is short-lasting, often not extending beyond 2 days. A modification of the procedures allowed us to load the cuffs with up to fifteen times the amount of TTX indicated above, drastically lowering the toxicity and lengthening the duration of the block. The key to the modification is to divide the calculated amount of NaCl into two parts: one is lyophilized with TTX, the other is lyophilized alone, then the two aliquots are mixed together with the silicone. The ratio NaCl lyophilized with TTX to NaCl lyophilized alone is not critical; we used 5 :1. This procedure was found accidentally and, although extremely effective, the reasons for the success are not entirely clear. A cuff containing fifteen times the amount used by Lavoie et al. (1977) (0-02 % x 15 = 0-3 %O) produces a total conduction block which lasts about 2 weeks. Smaller amounts of TTX (3 x, 5 x ) have been used in this study to obtain conduction blocks lasting 2-56 days. Completeness of the block was documented by the absence of twitch and tetanic contractions of e.d.l., soleus and other leg muscles, following stimulation of the ipsilateral sciatic nerve proximal to the cuff. Distal stimulation, on the other hand, evoked vigorous contractions and, in electrophysiological experiments, transmitted action potentials in all penetrated fibres. Electrophysiological techniques Soleus and/or e.d.l. muscles were dissected free and placed in a Plexiglas recording chamber containing 25 ml perfusion solution with the following composition (mm) NaCl, 135; KCl, 5; CaCl2, 2; MgCl2. 6H20, 1; NaHCO3, 15; Na2HPO4, 1; D-glucose, 1 1. The fluid was continuously recirculated in a closed system by bubbling a gas mixture (95 %O2, 5 % C02). The closed system had the advantage that smaller amounts of TTX were used during the experiments, making them less costly. Temperature was maintained at 28+1 C and ph was The extrajunctional membrane changes measured were the development of sensitivity to ACh and of resistance to TTX; in many experiments both were measured in the same region of each muscle fibre. A given fibre was penetrated with two micropipettes, one for recording voltage and the other for passing current; they were filled with 3 M-KCl and 2 M-K citrate, respectively, and placed /Zm apart. Sensitivity to micro-ionophoretically applied ACh was then measured in the region between the two electrodes using a third micropipette, filled with 3 M-AChCl, placed in a just-extracellular position; the resting membrane potential (r.m.p.) was set to -80 mv with the current pipette. ACh pipettes were several hundred megaohms in resistance and required less than 2 na braking current. ACh sensitivity is expressed in mv depolarization per 10-9 coulombs of charge passed through the pipette. A test muscle, denervated several days earlier, was always present in the chamber to check the ACh pipette properties. Often the same ACh pipette could be used for several days (kept at 4 C in between experiments) thus reducing variability in the measurements. Then, in order to test the resistance to TTX (10-6 M) of the action potential (Redfern & Thesleff, 1971), the r.m.p. was set to -100 mv and 10 ms depolarizing pulses were applied to exceed the threshold for spike

5 DENERVATION-INDUCED CHANGES IN MUSCLE 349 initiation. The spike was evoked at a constant latency of 4 ms and resistance to TTX was measured as the maximal rate of rise of the action potential given by its first derivative recorded with an RC circuit (100 kil, 100 pf). In partially denervated muscles, each fibre was labelled as innervated or denervated, depending on whether m.e.p.p.s were present or absent at the end-plate region. In the routine experiment on these muscles, resistance to TTX was measured at the end-plate region, and, in conjunction with sensitivity to ACh, 15-2 mm away from the end-plate: to ensure that one was in the same fibre, hyperpolarizing pulses from the current electrode were used as markers. When ACh sensitivity and TTX resistance were measured in muscles with a transplanted foreign nerve, this nerve was removed under the dissecting microscope immediately before the measurements. When a large number of measurements had to be taken, only TTX resistance was determined, which involves easier procedures than ACh micro-ionophoresis. In the many experiments in which both measurements were taken, there was an excellent correlation between their values (see, for example, Fig. 1 D-F and C-I). RESULTS Effect of partial denervation Partial denervation with innervated fibres normally active. It is known that after denervation, directly elicited muscle action potentials become resistant to the blocking action of TTX (Redfern & Thesleff, 1971). Partial denervation (see Methods) induced in soleus and e.d.l. muscles of the ipsilateral side the appearance of TTX-resistant action potentials not only in the denervated but also in the innervated fibres: measurements were made early after radicular nerve section (50-72 h) in the extrajunctional region close to the neuromuscular junction, as it is known that TTX resistance first appears in this region (Redfern & Thesleff, 1971). Fig. 1 A-C illustrates data pooled from normal and partially denervated soleus muscles. Normal muscles, represented in A, lack resistance to TTX since none of their fibres exhibit an action potential in the presence of a concentration of the poison of 10-6 M. In this respect the fibres of normal soleus and e.d.l. muscles differ from those of normal diaphragm, in which a moderate resistance to TTX has been detected in the end-plate region (Thesleff, Vyskocil & Ward, 1974; Cangiano & Lutzemberger, 1980). In the partially denervated muscles, all denervated fibres (Fig. 1 C) display well-developed action potentials in the presence of TTX, as expected. Fig. 1 B shows that some TTX resistance also develops in the innervated fibres, although to a much smaller extent than in the denervated ones. The effects of partial denervation in e.d.l. muscles are comparable to those observed in soleus (Cangiano & Lutzemberger, 1977). In three sham-operated animals in which the radicular nerve L5 was isolated but not sectioned, no TTX-resistant action potentials could be detected in either e.d.l. or soleus muscle. Furthermore, no TTX-resistant action potentials were found in normal e.d.l. and soleus muscles contralateral to the side of partial denervation. A reduction of action potential discharge in the remaining intact motoneurones cannot account for the moderate TTX resistance of the innervated fibres, because even total muscle inactivity fails to produce, at the short delay times of these experiments, any TTX resistance in most of the fibres (compare Fig. 1 D, hatched columns with Fig. 1 B). Partial denervation combined with paralysis of the innervated fibres. Since activity is known to exert a suppressive influence on extrajunctional ACh sensitivity (L0mo & Westgaard, 1976), ACh receptor synthesis (Hall & Reiness, 1977) and other membrane changes induced by denervation (L0mo & Westgaard, 1976), it seems

6 350 A. CANGIANO AND OTHERS reasonable to explain the small size of the effect induced by partial denervation in the innervated fibres, with the inhibitory influence of activity. Therefore, partial denervation was combined with chronic muscle paralysis by blocking nerve conduction at the time of radicular section (see Methods). Soleus and e.d.l. muscles were examined for the development of extrajunctional ACh sensitivity (1-52 mm from end-plates) Normal activity Paralysed Paralysed A D G ~~ LA ~ ~ C F 100 I I a I,, B E H _Xil--L okll a, ~~~~~~~~~~~~~~~10- Of ~~~ C ~~~~~F 0".~ ax raeo ieo pkelvsl H~~ oo Max. rate of rise of spikes (V s-'i Sensitivity to ACh (mv nc-') Fig. 1. Effects of partial denervation (56-72 h after L5 radicular nerve section) on resistance to TTX (A-F) and extrajunctional ACh sensitivity (G-I) of soleus muscles, in the absence (A-C) or in the presence (D-I) of TTX conduction block of sciatic nerve. A, normal muscles (five muscles, fifty fibres). B and C, innervated and denervated fibres, respectively, of partially denervated muscles (three muscles, twenty-eight innervated and twenty-two denervated fibres). D, hatched columns, purely paralysed muscles (five muscles, forty-five fibres); open columns, totally denervated muscles, (four muscles, thirty-five fibres). E and F, innervated paralysed, and denervated fibres, respectively, of partially denervated muscles with nerve conduction block (six muscles, thirty-three innervated and forty denervated fibres)., H and I, same muscles and fibres as D, E and F, respectively, but tested for ACh sensitivity. and TTX resistance, days after partial denervation. The amount of the changes observed in the innervated fibres was striking (Fig. 1 E) since it was as pronounced as in the denervated fibres of the same muscles (Fig. 1 F) and of totally denervated muscles (Fig. 1 D, open columns). The magnitude of the changes in the innervated fibres is best appreciated when compared to those of control, purely inactive muscles for the same length of time, in which no nerve degeneration has occurred: it is seen

7 DENER VATION-IND UCED CHANGES IN MUSCLE that in the latter, almost no muscle fibres have yet developed TTX resistance (Fig. 1 D, hatched columns). Similar behaviour was observed in the development of sensitivity to ACh, which was measured, along with TTX resistance, in the same extrajunctional regions. The data are presented in Fig. 1 G-I, displaying the same soleus muscle groups and fibres illustrated in D-F respectively. The effects of partial denervation on the inactive e.d.l. muscles were similar to those shown for the inactive soleus. Innervated inactive fibres had as high values of TTX resistance as denervated fibres, in three muscles ( V s-1, mean + S.E. of mean, twenty-two fibres and V s-1, sixteen fibres, respectively). By contrast, low values were found in paralysed control muscles with intact nerves ( V s-1 twenty-four fibres from three muscles). A question of interest is whether and to what extent reinnervation by collateral sprouting occurs in the partially denervated muscles. Freshly reinnervated fibres should have extrajunctional membrane changes because they belong initially to the denervated population, and should be discarded when looking at the effects of partial denervation on the innervated fibres. A simple approach to this problem is to measure in each innervated fibre the frequency of m.e.p.p.s, as it is known to be very low in freshly formed neuromuscular junctions (Bennett, McLachlan & Taylor, 1973; McArdle & Albuquerque, 1973). Fibres with abnormally low m.e.p.p. frequency (range s'1) were indeed recorded in partially denervated muscles that were not paralysed, although their incidence was very small (7 % of all innervated fibres). This is hardly surprising, given the short time after partial denervation (2-3 days) at which the observations were made. On the other hand, in the muscles in which partial denervation was combined with inactivity of the innervated fibres, abnormally low m.e.p.p. frequencies were not encountered. For example, the frequency range was s-1 (mean S.E. of mean) in thirty-one innervated inactive fibres of four partially denervated soleus as compared to s'l (mean S.E. of mean) in twenty-nine fibres of three purely paralysed soleus. This possibly indicates that very little reinnervation by collateral sprouting occurred in the denervated fibres (at least at the short delay time examined), perhaps due to the fact that the membrane changes are in this situation of equal intensity in the innervated inactive and the denervated fibres. Effects of varying the degree of partial denervation. Fig. 1 D-I shows that changes in membrane properties induced by partial denervation (both denervated and innervated inactive fibres) are quantitatively comparable to those induced by total denervation. The questions arose, therefore, whether one could find conditions in which a dependency of membrane effects on the degree of partial denervation was detectable and, if so, whether the innervated fibres would still be as equally affected as the denervated ones. To answer these questions, we examined the effects of partial denervation as early as 50 h, using muscles in which the percentage of denervated fibres varied greatly (7-94 % of all fibres recorded, see Methods). In these experiments a positive correlation was clearly apparent between the degree of partial denervation and the amount of TTX resistance, for both denervated (Fig. 2A) and innervated fibres (Fig. 2 B). Fig. 2 also shows that the magnitude of TTX resistance was virtually 351

8 352 A. CANGIANO AND OTHERS the same in the innervated and the denervated fibres of each partially denervated muscle, irrespective of the amount of denervation. Time of onset of membrane changes in innervated vs. denervated fibres. The identity of effects at 50 h in denervated and innervated inactive fibres at each level of TTX resistance suggests that the time of onset of the changes in the two classes of fibres A B >~~~~~~~~~ 100 0) L I *. p Percentage of denervated fibres Fig. 2. Effects of varying the degree of partial denervation (50 h) on resistance to TTX in A, denervated and B, innervated inactive fibres of soleus muscles. Control muscles are totally denervated (filled circles) and purely paralysed muscles (filled square, four muscles) at 50 h. Each point represents mean+s.e. of mean of values from four to fifteen fibres in a given muscle. The smallest number represents either denervated fibres in muscles with the lowest degree of denervation or innervated fibres in muscles with the highest degree of denervation. Regression lines, by the least-squares method, are shown; the correlation coefficient is 091 for the innervated and 0-87 for the denervated fibres; the angular coefficients of the two regression lines are comparable: 1-08 (innervated) and 1 10 (denervated). is similar. In order to ascertain whether the onset was indeed synchronous, a detailed time-course study was necessary. However, a delay of almost 2 days, which is characteristic of denervation-induced changes in rat muscles, might be too long to resolve a small difference in onset, say of a few hours. To optimize the time resolution, we sought an approach that could considerably shorten the latency of membrane changes after partial denervation. This was obtained by pre-paralysing the soleus muscle, through a conduction block of the sciatic nerve, and by performing 36 h later the partial denervation (see Methods for details). Fig. 3 illustrates that, under similar circumstances (1) TTX resistance appears with a latency much shorter than normal, (2) at 18 h after partial denervation (Fig. 3A and B) TTX resistance is just beginning to develop, with some fibres still normal and others with moderate resistance, (3) at 24 h the resistance appears slightly more pronounced (Fig. 3C and D), (4) both at 18 and 24 h no substantial difference between innervated (A, C) and denervated fibres (B, D) is detectable. The equality of effects in innervated and denervated fibres cannot be accounted for by trauma of the muscle inadvertently produced, even though the muscle was not exposed, at the time of partial section of the soleus

9 DENERVATION-INDUCED CHANGES IN MUSCLE nerve (see Methods). In fact in three animals the attempted section failed to damage any motor axon and in these cases no TTX resistance developed, in spite of the enhanced reactivity of the muscle due to chronic paralysis. Spread of effects from the site of nerve degeneration. It has been shown above that after partial denervation ACh sensitivity and TTX resistance develop to the same A 50 C.0 5 I I I L 0~~~~ D Max. rate of rise of spikes (V s-') Fig. 3. Synchrony in onset of TTX resistance near the end-plate region in innervated paralysed and denervated fibres of partially denervated soleus muscles. Muscles are first paralysed and 36 h later partially denervated. A, innervated and B, denervated fibres (fifty-three and sixteen, respectively) of four muscles tested 18 h after partial denervation. C, innervated and D, denervated fibres (nineteen and eighteen, respectively) of two muscles at 24 h. Thus, the over-all time after onset of paralysis is 54 (A, B) and 60 h (C, D). Purely paralysed control muscles for the same times do not exhibit TTX resistance (not shown). extent in denervated and innervated inactive fibres as measured in extrajunctional regions mm from the end-plates. Rat soleus and e.d.l. muscle fibres, on the other hand, are several millimetres long and after transaction of the muscle nerve the entire extrajunctional region develops ACh receptors and TTX-resistant action potentials. It is therefore of interest to ask if after partial denervation in inactive muscles (1) a similar spread of membrane changes occurs, and (2) the size of the changes in the innervated fibres remains as high as in the denervated fibres also in regions distant from end-plates. In partially denervated soleus muscles with chronic conduction block of the surviving axons, TTX resistance was determined by inserting micro- 12 PHY 355

10 354 A. CANGIANO AND OTHERS electrodes at regular intervals in denervated and innervated inactive fibres from the end-plate to myotendinous region. A representative experiment is illustrated in Fig. 4 which shows that (1) TTX resistance is similar from end-plate to tendon in an innervated inactive (open triangles) and a denervated fibre (open circles) of a partially denervated soleus at 72 h, and (2) the changes are also well developed in distant 200 r -._ VI 4_ 0 10O0 4) 0 0' 0 Region near end-plates I 3 6 I 9 Tendon region Distance (mm) Fig. 4. Resistance to 1TX measured along individual fibres ofsoleus muscles from end-plate to tendon. Open triangles, innervated inactive fibre and open circles, denervated fibre from a partially denervated paralysed muscle. Filled circles, denervated fibre from a totally denervated muscle; filled triangles, fibre from a purely paralysed muscle. All fibres examined at 72 h. regions although detectably smaller than in a fibre of a totally denervated muscle (filled circles). The last feature is not unexpected, due to the smaller amount of degenerating nerve tissue in the partially denervated than in the totally denervated muscle. Similar results were obtained from two other partially denervated muscles. Effects of degeneration of axons other than motor Sensory axons. Having observed the effects of partial denervation on innervated fibres, an obvious question arising is whether degeneration of only the sensory innervation of the muscle will induce extrajunctional ACh sensitivity and ITX resistance. Brown et al. (1978) have indeed observed the development of some supersensitivity to bath-applied ACh and of motor nerve sprouting after removal of the appropriate dorsal root ganglia in mouse muscles. Our experiments were directed to confirm and extend these findings to inactive muscles, as well as to quantify the effects in the rat.

11 DENERVATION-INDUCED CHANGES IN MUSCLE A x x E Region near Tendon E Region near Tendon end-plates region end-plates region Distance (mm) 100Distance (mm) D 60 L c.0~~~~~~~~~~ 40-0( 50 - a, >1io Sensitivity to ACh (mv nc1i) Region near Tendon edplae Distance (mm) region Fig. 5. Effects of degeneration of axons other than motor. A, effects of transection of sensory axons (ablation of dorsal root ganglia L4 and L5) on TTX resistance of chronically inactive coleus muscles, at 3-5 days. Filled circles, six deafferented muscles. Open circles, nine purely paralysed muscles. Each point is the mean + 5.E. ofmean of values from fibres. The dashed line represents the lowest level of TTX resistance usually found in denervated muscles at comparable times. B and C, membrane changes in soleus muscles following efferent denervation (section of ventral roots) and total denervation (efferent plus afferent, by section of radicular nerves) at 48 h. B, TTX resistance in five muscles with efferent denervation (open circles) and five muscles with total denervation (filled circles). Values from twenty to fifty fibres. Dashed line as in A. C, extrajunctional ACh sensitivity at 3 mm from end-plates, three muscles with efferent denervation (open columns, thirty-six fibres) and three muscles with total denervation (filled columns, thirty-five fibres). Nerve stump length was the same with efferent and total denervation. D, lack of effects of sympathetic denervation on TTX resistance in normally active (two muscles, lower graph) and chronically paralysed (four muscles, upper graph), at 3-5 days. In both graphs: open circles, control muscles; filled circles, experimental muscles. Values from fifteen to thirty fibres. Dashed line as in A. Our first approach was to remove L4 and L5 dorsal root ganglia and look for the development of TTX resistance and ACh sensitivity in extrajunctional regions of the ipsilateral soleus muscle after 3-5 days. At the beginning of the acute micro-electrode experiment, sixty to eighty fibres were recorded at the end-plate region to make sure that no denervated fibres were present due to accidental damage to ventral roots. Muscles in which not all fibres had m.e.p.p.s were discarded. Since in preliminary experiments no extrajunctional membrane changes were detected, we combined deafferentation with conduction block of the ipsilateral sciatic nerve, to produce chronic inactivity of the coleus muscle. Fig. 5A shows that TTX resistance at about 12-2

12 356 A. CANGIANO AND OTHERS 3 days was significantly greater in the deafferented inactive muscles (filled circles) than in control, purely inactive muscles (open circles). It is also apparent that, in absolute terms, the TTX resistance is not very pronounced, the dashed line representing the lowest level usually found in denervated muscles, at comparable times. Instead, innervated inactive fibres of partially denervated muscles have values above this level. It is unlikely that even a low level of partial denervation existed in the deafferented muscles since all fibres recorded had m.e.p.p.s. Nevertheless, in order to be certain that the observed effects were genuinely due to degeneration of sensory axons, we resorted to a different approach. If degeneration of these axons contributes to the onset of membrane changes following denervation, cutting both efferent and afferent innervation of muscle ('total denervation') should produce greater effects than cutting the efferent innervation alone (see Methods), particularly at early times, i.e. before saturation of effects can occur. Fig. 5B and C shows that this was indeed the case for both TTX resistance (B) and extrajunctional ACh sensitivity (C). Sympathetic axons. We next investigated the effect of sectioning the sympathetic innervation of soleus muscle, by removing bilaterally the lumbar sympathetic chain. Fig. 5D indicates that this procedure was completely ineffective, both in normally active soleus muscles (lower graph) and in functionally inactivated ones (upper graph), with regard to TTX resistance. The same was true for extrajunctional ACh sensitivity (not shown), up to 7 days after sympathetic denervation. Degeneration of a foreign nerve Fibular nerve. All experimental situations presented above have in common (1) that degenerating nerve tissue is produced in the vicinity of innervated muscle fibres, either normally active or paralysed, and (2) that, in order to obtain this result, interference is made with the normal innervation of muscle, whether motor, sensory or autonomic. It was then of interest to investigate the effects of degenerating nerve tissue derived from foreign nerves not making synapses with the muscle, so that no reduction of the over-all muscle innervation was produced. A transplanted muscle nerve, the superficial fibular, was allowed to grow close to the fibres of soleus muscle (Fex & Thesleff, 1967) for several weeks, in an extrajunctional region. No synapses were made, at any stage, because the original soleus nerve was left intact. Nerve breakdown products were then made to appear on the surface of the soleus muscle fibres, by sectioning the transplanted nerve. The muscle was either normally active or paralysed at the time of foreign nerve section with the usual conduction block of the sciatic nerve. A few days later (2 S5-) development of ACh sensitivity and TTX resistance was assessed under the degenerating foreign nerve as well as in distant regions. Examples of the results obtained are illustrated in Figs. 6, 7 and 8B. Fig. 6 illustrates the results obtained from four differently treated soleus muscles and shows that (a) when the foreign nerve was sectioned and the soleus muscle was normally active no resistance to TTX developed (filled triangles); (b) when, however, section of the foreign nerve was combined with chronic inactivity of the muscle (filled circles), a peak of TTX resistance appeared under the degenerating nerve, reaching levels similar to those following normal denervation, i.e. after section of the original soleus nerve (open triangles); (c) paralysis alone (open circles) only produced a low

13 DENERVATION-INDUCED CHANGES IN MUSCLE 357 level of TTX resistance, illustrating the longer delay of onset of membrane changes after functional inactivation vs. denervation of muscle. It should also be noted that in the purely paralysed muscle (open circles) a transplanted nerve was also present but not sectioned, confirming that section of this nerve is a necessary step in the production of the peak of TTX resistance. Fig. 7 illustrates the results from another group of four soleus muscles in which sensitivity to ACh was measured. The muscles were treated in the same way and are CD co /T S_ \ t Region near Foreign Tendon end-plates nerves region Distance (mm) Fig. 6. Effects on resistance to TTX of soleus muscles induced by section of a transplanted superficial fibular nerve, at h. Filled triangles, section of transplanted nerve but soleus muscle normally active. Filled circles, section of transplanted nerve combined with muscle inactivity by chronic conduction block of sciatic nerve. Control muscles are: open triangles, totally denervated soleus muscle; open circles, purely paralysed muscle. Each point represents mean + S.E. of mean of ten to twelve adjacent fibres in one muscle. identified with the same symbols as in Fig. 6. It is seen that the results are consistent with those shown in Fig. 6 for TTX resistance. The filled circles in Fig. 7 show the results from a muscle that was both paralysed and had the foreign nerve sectioned; there is a clear increase in the sensitivity of the extrajunctional membrane to ACh, particularly at the site of the degenerating foreign nerve. Resistance to TTX was also measured in this muscle, in the same fibres and regions where ACh sensitivity was assessed, and the results are shown in Fig. 8B, filled circles. Sural nerve. The superficial fibular is a muscle nerve, containing both efferent and afferent axons. By transplanting a sensory nerve, such as the sural, on the soleus muscle one can test the effect of breakdown products of afferent axons in isolation (see Methods for details). This was done in three animals in which degeneration of foreign axons was combined, as usual, with soleus chronic paralysis. All three cases showed development of TTX resistance under the degenerating sural nerve, which was, however, significantly less pronounced than under the degenerating fibular nerves, at comparable times. An example of sural nerve degeneration is shown in Fig. 8A, while

14 358 A. CANGIANO AND OTHERS Fig. 8B, filled circles, illustrates an example of fibular nerve degeneration, in another muscle, for comparison. An approximate estimate of the difference in effectiveness between sensory and mixed nerve degeneration can be obtained by pooling together the peak values of TTX resistance measured under the degenerating nerves in all muscles examined, with the following results: sural, V s-i (three muscles, thirty-six 200 L u c E 4- Ca 100-.A T -I I.,L_ c F 01 I 50 OL Foreign nerve 0 2 t Regi on near Foreign Ten end -plates nerves regi idon lion Distance (mm) Fig. 7. Effects on extrajunctional ACh sensitivity of soleus muscles induced by section of a transplanted superficial fibular nerve, at h. Meaning of symbols as in Fig. 6. Each point represents mean + s.e. of mean of seven to twelve adjacent fibres in one muscle. fibres); fibular, V s-' (five muscles, thirty-nine fibres). These results strongly suggest that degenerating motor axons are substantially more effective than sensory ones in inducing membrane changes in skeletal muscle. The difference might actually be larger than the above figures suggest, for the following reasons: (1) the superficial fibular nerve contains both efferent and afferent axons, (2) the sural is a nerve somewhat bigger than the fibular, as could be clearly detected in the dissecting microscope in the region of the nerve outgrowth on the muscle, (3) some effect is also caused by paralysis alone and thus has a larger share in the smaller effect of the sural, and (4) when degeneration of foreign sensory axons is induced by removal of dorsal root ganglia (as in Fig. 8A), part of the effect may be due to degeneration of the muscle's own sensory fibres. Effects of dexamethasone. An attempt was made to test if an inflammation-like

15 DENER VATION-INDUCED CHANGES IN MUSCLE A B ID L 0 2 4t t 6 8 Region near Foreign Tendon Region near Foreign Tendon end-plates nerve region end-plates nerve region Distance (mm) Fig. 8. A illustrates the effects on TTX resistance induced by section of foreign sensory axons in a chronically paralysed soleus muscle, at 90 h. Foreign nerve is the sural and deafferentation is obtained by removal of dorsal root ganglia L4 and L5. Each point represents mean + S.E. of mean of eight to ten adjacent fibres. In B, filled circles, the effects on TTX resistance of sectioning a transplanted mixed nerve (superficial fibular) in a different soleus muscle, are shown, for comparison. Same muscle and fibres as shown in Fig. 7, filled circles, for ACh sensitivity measurements. Control muscles, one totally denervated (filled triangles) and one purely paralysed (open triangles), are taken from Fig. 6. 0L TABLE 1. Lack of effect of dexamethasone on TTX resistance and ACh sensitivity induced in paralysed soleus muscles by section of transplanted fibular nerves Resistance to TTX (max. rate of rise of action potentials; V s-1) Sensitivity to ACh (mv nc-1) Untreated Dexamethasone Untreated Dexamethasone Under the foreign nerve (5, 39) (4, 28) (1, 7) (1, 8) 2 mm from foreign nerve (5, 36) (4, 20) The values shown represent mean + S.E. of mean of measurements made under the degenerating foreign nerve and about 2 mm from its border. First and second numbers in parentheses represent the number of muscles and fibres, respectively. Time, about 3 days after section of foreign nerve. reaction associated with nerve degeneration is responsible, at least in part, for the muscle membrane changes observed following section of foreign nerves. For this purpose we repeated the experiment of cutting transplanted fibular nerves in paralysed soleus muscles, in animals treated with substantial doses of an antiinflammatory agent such as dexamethasone (see Methods). The drug treatment did not induce any modification, with respect to untreated animals, either in peak amplitude or in spatial extent of both membrane changes, TTX resistance and ACh

16 360 A. CANGIANO AND OTHERS sensitivity (Table 1). In another group of control animals, dexamethasone did not induce any membrane change in normal soleus muscles (two), muscles with section of a transplanted nerve but normally active (two), and did not affect the high level of TTX resistance and ACh sensitivity characteristic of 3-4 days denervated soleus (two muscles). DISCUSSION Three main results derive from the present experiments: (1) normally innervated muscle fibres develop ACh supersensitivity and TTX resistance after section and peripheral degeneration of original and transplanted nerve fibres in the same muscle; (2) inactivity induced by TTX conduction block of intact axons strongly potentiates the response of muscle to adjacent nerve fibres degeneration; (3) destruction of motor axons is a much more potent stimulus than destruction of sensory or sympathetic axons. Partial denervation The experiments show that partial denervation affects the membrane properties of denervated as well as innervated fibres. Innervated active fibres respond weakly to partial denervation whereas fibres inactivated by TTX conduction block of the nerve, respond as strongly as denervated fibres: i.e. intact innervation with maintained axonal transport (Lavoie et al. 1976; Pestronk et al. 1976) and spontaneous ACh release cannot prevent full-blown denervation-like changes from developing. One deals with a potentiation and not with a mere summation of effects because inactivity alone has negligible effects at early times (see Fig. 1D and G). This probably explains why the response to inactivity alone was over-estimated in early experiments (L0mo & Rosenthal, 1972; Cangiano et al. 1975; Cangiano et al. 1977): in these experiments conduction block was induced by local compression of the nerve which almost invariably causes some degree of denervation. The potentiating action of inactivity makes the effect of partial denervation so clear-cut that any doubt raised (Tiedt, Albuquerque & Guth, 1977) about the efficacy of partial denervation in inducing membrane changes in the innervated fibres is overcome. It should be emphasized that the extrajunctional effects in innervated inactive fibres are equal in size to those in denervated fibres, both close and far from end-plates (see Figs. 1 and 4, respectively). This was found to be true also when the amount of membrane changes was small, due to a small degree of partial denervation (Fig. 2). The straightforward interpretation is that nerve degeneration causes substances to appear in the interstitium which act on all fibres in that region, and that these fibres are equally responsive, whether denervated or innervated, because they are all inactive. In support of this interpretation is the finding that innervated inactive and denervated fibres are synchronous in time of onset of their membrane changes, even when their latency is made very short (about 18 h, Fig. 3), to optimize the time resolution. In fact the synchrony in onset makes unlikely the possibility that the changes in innervated fibres are secondary to those in the denervated fibres, for in this case the onset should be delayed in the innervated fibres. The synchrony in onset

17 DENERVATION-INDUCED CHANGES IN MUSCLE also argues against the possibility that the changes induced in the innervated fibres by partial denervation are due to retrograde effects - through the spinal cord - on intact motoneurones, leading to impaired supply of some 'trophic' regulatory substance. The peripheral mechanism of action of partial denervation is further supported by the finding that if partial denervation is made closer to the muscle the membrane changes appear sooner in both denervated and innervated fibres (Cangiano & Lutzemberger, 1980). Section of transplanted fibular nerves The main result of sectioning a non-innervating, transplanted mixed nerve such as the superficial fibular is similar to that observed in the innervated fibres following partial denervation, i.e. full-blown ACh supersensitivity and TTX resistance develop, provided the muscle has been chronically paralysed. This demonstrates how sensitive inactive muscle fibres become despite intact innervation. Again, the straightforward interpretation of the effects is based on breakdown products of the nerve, since the transplanted nerve has always remained foreign to the muscle and the effects are localized under the degenerating axons. Two features, however, distinguish the muscle reaction to degeneration of a foreign nerve from that to partial denervation. First, degeneration of the foreign nerve alone, i.e. without muscle paralysis, is ineffective whereas partial denervation produces some effect in the innervated fibres even when they are active. Secondly, the membrane changes following transaction of a foreign nerve are prominent under the nerve but strongly attenuate with distance, whereas partial denervation produces changes in the entire muscle. Both differences indicate a smaller efficacy of foreign nerve transaction with respect to partial denervation. This could be explained by the fact that, since the foreign nerve does not make synapses, its breakdown products are released at a greater distance from the muscle. On the other hand, the stimulus from a degenerating transplanted nerve becomes much more potent if it makes synapses before it is cut. In that case a marked supersensitivity develops even in innervated active soleus fibres. This has been shown to occur near the denervated foreign end-plates of soleus muscles dually innervated (L0mo & Westgaard, 1976). The effect may be related not only to the shorter distance between degenerating nerve terminals and muscle fibres, but also to larger amounts of inducing factors from differentiated terminals, particularly from ectopic terminals which are multiple and unusually large (T. L0mo & E. Waerhaug, personal communilation). This interpretation may also apply to the effect of partial denervation on innervated active fibres. The absence of effects following breakdown of a non-innervating transplanted nerve, unless combined with muscle inactivity, seems different from the ACh supersensitivity developing after application of a piece of nerve on the surface of normally active muscle (Jones & Vrbova, 1974; Jones & Vyskocyl, 1975). However, it is known that trauma (Katz & Miledi, 1964) and a foreign body (Jones & Vrbova', 1974) can themselves induce ACh supersensitivity. A potentiating action from these factors can thus explain the ability of a piece of nerve, or of nerve extracts embedded in silastic plates (Vyskocil, Syrovy & Prusik, 1981), to induce ACh supersensitivity even in active muscle. Consistent with this interpretation is the report that segments of nerves implanted on the muscle surface produce an inflammatory reaction with 361

18 362 A. CANGIANO AND OTHERS presence of degenerating and regenerating muscle fibres (Guth, Richman, Barrett, Warnick & Albuquerque, 1980), while no such features have been observed near the degenerating intramuscular nerves after denervation (Murray & Robbins, 1982). Section of sensory and sympathetic nerves The effects of deafferentation (see Fig. 5A-C) confirm and extend those shown in the mouse by Brown et al. (1978) and support the notion that nerve breakdown products are able to affect the membrane properties of muscle fibres. The small size of these effects, on the other hand, suggests that different types of axons are not equally effective, the motor ones being the most effective. Consistent with this idea is the absence of any effect following degeneration of sympathetic axons despite the potentiating action of chronic inactivity (see Fig. 5 D). The hypothesis of a hierarchy among fibre types is also strongly supported by the finding that degeneration of a transplanted sensory nerve (sural, see Fig. 8A) produces much smaller changes in muscle membrane properties than does degeneration of a mixed nerve (superficial fibular, Figs. 6, 7 and 8B). Finally, a correlation appears to exist between these findings and the observation that sensory and sympathetic neurones, unlike motoneurones, lack the ability to aggregate ACh receptors in the membrane of muscle cells in culture (Cohen & Weldon, 1980). Possible mechanisms and implications It is useful to quantitatively evaluate the response of inactive membranes to nerve breakdown products, with reference to the effects of inactivity alone and to those of denervation. At early times, such as those selected for the present experiments, a large gap exists between the negligible effects of pure inactivity and the almost maximal effects of denervation. This gap is completely bridged by the effect of nerve destruction on inactive membranes, as seen locally with the foreign nerve paradigm, and over the entire end-plates-tendon region with the partial denervation paradigm. Thus, the greater efficacy of denervation vs. pure inactivity can be well explained by nerve breakdown products acting diffusely throughout the muscle on membranes highly reactive because of the concomitant muscle inactivity. Effects related to removal of ACh or of other nerve-borne neural factors from denervated muscles (Miledi, 1960), particularly in preparations near the end-plates (Bray et al. 1982), may be additional mechanisms explaining the greater efficacy of denervation with respect to inactivity alone. For example, despite the difficulties in maintaining a prolonged and complete paralysis, there is evidence for a long-term difference in membrane properties between denervated and paralysed muscles (Gilliatt et al. 1978; Bray, Hubbard & Mills, 1979; see however, Stanley & Drachman, 1979); a straightforward interpretation of these findings is based on the persistence of some neural regulatory influence in the paralysed muscles. Furthermore, experiments producing muscle paralysis by blocking ACh receptors with a-bungarotoxin have shown larger ACh supersensitivity and TTX resistance than in nerve conduction block experiments, indicating a role for ACh as a neurotrophic transmitter (Mathers & Thesleff, 1978). In subsequent experiments, the effects of a-bungarotoxin have been potentiated, through chronic direct application on the muscle, to become as large as those of denervation (Drachman et al. 1982). However, the increased responsiveness

19 DENERVATION-INDUCED CHANGES IN MUSCLE of inactive fibres demonstrated in the present experiments, points to the need for caution in interpreting data obtained from muscles that are paralysed; for example, muscles controlling the effects of trauma involved in chronic drug application should also be paralysed (Drachman et al. 1982). The nature of nerve breakdown products and their mechanism of action is unknown. An indirect action on muscle fibres through an inflammatory reaction is unlikely to be the main mechanism, as suggested by the facts that (a) degeneration of a transplanted sensory nerve is much less effective than that of a mixed nerve, and (b) dexamethasone did not reduce significantly the effects of sectioning transplanted fibular nerves (see Table 1). Consistent with these observations is, on the other hand, the possibility that nerve breakdown products induce the aggregation of ACh receptors and other membrane changes in the extrajunctional membrane through direct action of some factor normally contained in axons, especially the motor ones. Normal motor nerves have the ability to concentrate the ACh receptors in the muscle membrane (for a review, see Fambrough, 1979), as occurs during development when the new nerve-muscle contacts are established. Therefore, it seems reasonable to suggest that a common chemical physiological factor is shared by this junctional effect and the action of nerve breakdown products. When an adult muscle is denervated, due to breakdown of the nerve, the ACh receptor-aggregating factor could be released in the interstitium between the muscle fibres, thus becoming able to act at a distance. The high efficacy could be explained by the concomitant inactivity that makes the extrajunctional membrane much more responsive. Although this particular interpretation of the action of nerve breakdown products can account, together with inactivity, for the appearance of extrajunctional ACh receptors after denervation, supporting evidence must await future experimentation. 363 REFERENCES ALBUQUERQUE, E. X., WARNICK, J. E., TASSE, J. R. & SANSONE, F. M. (1972). Effects of vinblastine and colchicine on neural regulation of the fast and slow skeletal muscles of the rat. Experimental Neurology 37, AXELSSON, J. & THESLEFF, S. (1959). A study of supersensitivity in denervated mammalian skeletal muscle. Journal of Physiology 147, BENNETT, M. R., MCLACHLAN, E. M. & TAYLOR, R. S. (1973). The formation of synapses in reinnervated mammalian striated muscle. Journal of Physiology 233, BETZ, W. J., CALDWELL, J. H. & RIBOHESTER, R. R. (1980). The effects of partial denervation at birth on the development of muscle fibres and motor units in rat lumbrical muscle. Journal of Physiology 303, BRAY, J. J., FORREST, J. W. & HUBBARD, J. I. (1982). Evidence for the role of non-quantal acetylcholine in the maintainance of the membrane potential of rat skeletal muscle. Journal of Physiology 326, BRAY, J. J., HUBBARD, J. I. & MILLS, R. G. (1979). The trophic influence of tetrodotoxin-inactive nerves on normal and reinnervated rat skeletal muscles. Journal of Physiology 297, BROWN, M. C., HOLLAND, R. L. & IRONTON, R. (1978). Degenerating nerve products affect innervated muscle fibres. Nature 275, BUCKLEY, G. A. & HEATON, J. (1968). A quantitative study of cholinesterase in myoneural junctions from rat and guinea-pig extraocular muscles. Journal of Physiology 199, CANGIANO, A. (1973). Acetylcholine supersensitivity: the role of neurotrophic factors. Brain Research 58, CANGIANO, A. & FRIED, J. A. (1977). The production of denervation-like changes in rat muscle by

20 364 A. CANGIANO AND OTHERS colchicine, without interference with axonal transport or muscle activity. Journal of Physiology 265, CANGIANO, A., L0Mo, T., LUTZEMBERGER, L. & SVEEN, 0. (1980). Effects of chronic nerve conduction block on formation of neuromuscular junctions and junctional AChE in the rat. Ada physiologica scandinavica 109, CANGIANO, A. & LUTZEMBERGER, L. (1977). Partial denervation affects both denervated and innervated fibers in the mammalian skeletal muscle. Science 196, CANGIANO, A. & LUTZEMBERGER, L. (1980). Partial denervation in inactive muscle affects innervated and denervated fibres equally. Nature 285, CANGIANO, A., LUTZEMBERGER, L. & NIcOTRA, L. (1977). Non-equivalence of impulse blockade and denervation in the production of membrane changes in rat skeletal muscle. Journal of Physiology 273, CANGIANO, A., LUTZEMBERGER, L. & ZORUB, D. S. (1975). Effects of inactivity on muscle membrane properties in rats. Neuroscience Abstracts 1, 766 (1178). COHEN, M. W. & WELDON, P. R. (1980). Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type. Journal of Cell Biology 86, DRACHMAN, D. B., STANLEY, E. F., PESTRONK, A., GRIFFIN, J. W. & PRICE, D. L. (1982). Neurotrophic regulation of two properties of skeletal muscle by impulse-dependent and spontaneous acetylcholine transmission. Journal of Neuroscience 2, FAMBROUGH, D. M. (1979). Control of acetylcholine receptors in skeletal muscle. Physiological Reviews 59, FEX, S. & THESLEFF, S. (1967). The time required for innervation of denervated muscle by nerve implants. Life Sciences 6, GILLIATT, R. W., WESTGAARD, R. H. & WILLIAMS, J. I. (1978). Acetylcholine sensitivity of denervated and inactivated baboon muscles. Journal of Physiology 280, GUTH, L., RICHMAN, E., BARRETT, C., WARNICK, J. E. & ALBUQUERQUE, E. X. (1980). The mechanism by which degenerating peripheral nerve produces extrajunctional acetylcholine sensitivity in mammalian skeletal muscle. Experimental Neurology 68, HALL, Z. W. & REINESS, C. G. (1977). Electrical stimulation of denervated muscles reduce incorporation of methionine into the ACh receptor. Nature 268, HARRIS, J. B. & THESLEFF, S. (1972). Nerve stump length and membrane changes in denervated skeletal muscle. Nature, New Biology 236, HOFMANN, W. W. & THESLEFF, S. (1972). Studies on the trophic influence of nerve on skeletal muscle. European Journal of Pharmacology 20, JONES, R. & VRBOVA, GERTA (1974). Two factors responsible for the development of denervation hypersensitivity. Journal of Physiology 236, JONES, R. & VYSKOIL, F. (1975). An electrophysiological examination of the changes in skeletal muscle fibres in response to degenerating nerve tissue. Brain Research 88, KATZ, B. & MILEDI, R. (1964). The development of acetylcholine sensitivity in nerve-free segments of skeletal muscle. Journal of Physiology 170, LAVOIE, P. A., COLLIER, B. & TENENHOUSE, A. (1976). Comparison of a-bungarotoxin binding to skeletal muscles after inactivity or denervation. Nature 260, LAVOIE, P. A., COLLIER, B. & TENENHOUSE, A. (1977). Role of skeletal muscle activity in the control of muscle acetylcholine sensitivity. Experimental Neurology 54, L0Mo, T. (1974). Neurotrophic control of colchicine effects on muscle? Nature 249, LoMo, T. & ROSENTHAL, J. (1972). Control of ACh sensitivity by muscle activity in the rat. Journal of Physiology 221, LoMO, T. & WESTGAARD, R. H. (1976). Control of ACh sensitivity in rat muscle fibres. Cold Spring Harbor Symposia on Quantitative Biology 40, McARDLE, J. & ALBUQUERQUE, E. X. (1973). A study of the reinnervation of fast and slow mammalian muscle. Journal of General Physiology 61, MATHERS, D. A. & THESLEFF, S. (1978). Studies on neurotrophic regulation of murine skeletal muscle. Journal of Physiology 282, MILEDI, R. (1960). The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation. Journal of Physiology 151, 1-23.

21 DENER VATION-IND UCED CHANGES IN MUSCLE 365 MILEDI, R. & SLATER, C. R. (1969). On the degeneration of rat neuromuscular junction after nerve section. Journal of Physiology 207, MURRAY, M. A. & ROBBINS, N. (1982). Cell proliferation in denervated muscle: identity and origin of dividing cells. Neuroscience 7, PESTRONK, A., DRACHMAN, D. B. & GRIFFIN, J. W. (1976). Effect of muscle disuse on acetylcholine receptors. Nature 260, PURVES, D. (1976). Long-term regulation in the vertebrate peripheral nervous system. International Review of Physiology and Neurophysiology II 10, REDFERN, P. & THESLEFF, S. (1971). Action potential generation in denervated rat skeletal muscle. II. The action of tetrodotoxin. Acta physiologica scandinavica 82, STANLEY, E. F. & DRACHMAN, D. B. (1979). Effects of disuse on the resting membrane potential of skeletal muscle. Experimental Neurology 64, THESLEFF, S., VYSKOI6L, F. & WARD, M. R. (1974). The action potential in end-plate and extrajunctional regions of rat skeletal muscle. Acta physiologica scandinavica 91, TIEDT, T. N., ALBUQUERQUE, E. X. & GUTH, L. (1977). Degenerating nerve fiber products do not alter physiological properties of adjacent innervated skeletal muscle fibers. Science 198, UCHITEL, 0. & ROBBINS, N. (1978). On the appearance of acetylcholine receptors in denervated rat diaphragm and its dependence on nerve stump length. Brain Research 153, VRBOVA, GERTA. (1967). Induction of an extrajunctional chemosensitive area in intact innervated muscle fibres. Journal of Physiology 191, 20-21P. VYSKO6In, F., SYROVY, I. & PRUSIK, Z. (1981). Induction of extrajunctional acetylcholine sensitivity of rat EDL muscle by peptidic component of peripheral nerve. PftAgers Archiv 390,