J. Physiol. (1983), 340, pp. 175-194 175With 7 text-ftgure8

Printed in Great Britain

LOCAL AND SYSTEMIC EFFECTS OF TETRODOTOXIN ON THEFORMATION AND ELIMINATION OF SYNAPSES IN REINNERVATED

ADULT RAT MUSCLE

BY TORFINN TAXTFrom the In8titutes of Phy8iology and Neurophy8iology, Karl Johans Gate 47,

Oslo 1, Norway

(Received 3 November 1982)

SUMMARY

1. Polyneuronal innervation of normal and reinnervated fourth deep lumbricalmuscle fibres was studied with tension measurements and intracellular recordings.From the tenth day after a complete crush of the muscle nerve, some of thereinnervated muscles were completely paralysed for up to 15 days by local applicationof tetrodotoxin (TTX) to the sciatic nerve. Other animals received only systemicinfusion of TTX during the muscle reinnervation.

2. Measurements oftetanic-tension overlap suggested that about 6% ofthe musclefibres in the normal lumbrical muscle were polyneuronally innervated, while intra-cellular recordings suggested that the percentage was as high as 25 %. This discrepancywas mainly due to the presence ofone small, sub-threshold end-plate potential (e.p.p.)and one large, suprathreshold e.p.p. in almost all polyneuronally innervated musclefibres.

3. Intracellular recordings during muscle reinnervation showed that the extent ofpolyneuronal innervation reached a maximum of 50% 10-15 days after denervationand that by 16-20 days this had decreased to a level similar to that found in normalmuscle.

4. After a week of total muscle paralysis the extent of polyneuronal innervationhad increased to about 80 %, estimated by both tension measurements and intra-cellular recordings. Subsequently, there was no sign of any net elimination of thepolyneuronal innervation, even in muscles paralysed for up to two weeks. Many ofthe polyneuronally innervated fibres were innervated by at least two motor axons.each producing suprathreshold e.p.p.s.

5. In muscles contralateral to the paralysed muscles, the extent of polyneuronalinnervation reached a maximum of 50% 10-15 days after denervation as inreinnervated muscles not exposed to TTX. But in contrast to the subsequent decreasein the extent of polyneuronal innervation in animals which received no TTX, thislevel of polyneuronal innervation persisted in muscles contralateral to the paralysedmuscles. The same was true for reinnervated muscles in animals which only receivedTTX systemically.

6. The increased level of polyneuronal innervation after TTX application was notcaused by differences in the number of motor units or in number of muscle fibres.

7. Paralysed muscles relaxed much more slowly than non-paralysed muscles at theend of a fused tetanic contraction. The tetanus/twitch ratio of these muscles was alsosmaller than in contralateral control muscles and the rise time of the twitch wasgreater.

8. It is concluded that a substantial fraction of the fibres in the normal lumbricalmuscle of young rats is polyneuronally innervated. After reinnervation, the normalinnervation pattern is re-established, but no net elimination of the polyneuronalinnervation occurs unless either nerve or muscle or both are active. A net eliminationofsynapses is also prevented when TTX is present systemically in low concentrations.

INTRODUCTION

There is convincing evidence that activity is one of the important factors whichdetermines the connectivity of the nervous system during development (see Harris,1981, for review). Much less is known about how activity may modify connectivityin the adult nervous system (Grinnell & Herrara, 1981). Due to its simple structureand accessibility, the neuromuscular system has been used extensively as a modelsystem to study how activity influences the formation and elimination of synapses.During the neonatal period when elimination of neuromuscular synapses normallyoccurs (Redfern, 1970; Brown, Jansen & Van Essen, 1976) muscle paralysis completelystops the elimination (Thompson, Kuffler & Jansen, 1979; Brown, Holland &Hopkins, 1981). The elimination of the polyneuronal innervation in adult skeletalmuscles that follows reinnervation (McArdle, 1975) is also changed by inactivityduring reinnervation (Benoit & Changeux, 1978). However, it is not clear whetherthis inactivity only decreases the rate of synapse elimination or prevents theelimination completely.To study this problem, one leg of adult rats was paralysed by local tetrodotoxin

(TTX) superfusion of the right sciatic nerve 10 days after a complete crush of thenerve to a lumbrical muscle on both sides. The block lasted up to 15 days. Someanimals with crushed muscle nerves received only continuous systemic infusion ofTTX, while others were used as controls with no TTX application. This experimentalarrangement also allowed a study of possible contralateral effects of the total nerveblock, and raised the possibility of systemic effect ofTTX in addition. The only effectofTTX reported previously is the reversible block of voltage-dependent Na channels(Moore, Blaustein, Anderson & Narahashi, 1967; Rogart, 1981). Because of this, TTXhas been used as a neurotoxin which selectively blocks activity in nerves andneurones. Such blocks were thought to allow studies of the exclusive influence ofactivity on the nervous system during development, in adults and following lesions(Brown & Ironton, 1977; Roper & Ko, 1978; Braithwaite & Harris, 1979; Thompsonet at. 1979; Betz, Caldwell & Ribehester, 1980; Harris, 1980).The results suggest that TTX has both local and systemic effects on the time course

and extent of the polyneuronal innervation and on the contractile properties ofreinnervated lumbrical muscle. Caution is therefore required before effects seen usingTTX are attributed solely to an effect on neuronal impulse activity.

176 T. TAXT

EFFECTS OF TTX ON SYNAPSES

METHODS

All rats were about 40 days of age and weighed 120-140 g at the start of the experiments. Theirweights at the time of the final experiments were 150-200 g. The rats were anaesthetized withsodium pentobarbitone (60 mg/100 g ofbody weight i.P.) during operations for chronic experimentsand with ether for acute experiments. Most of the normal and the experimental rats were kept incages with flat plastic bottoms. The remaining were kept in large cages with bottoms ofthick-gaugedwire screening.

The experiments were carried out using the fourth deep lumbrical muscle in the hind foot of rats.There were three stages: first, denervation of the muscles in both legs and the reinnervation bythe regenerating nerves; secondly, total nerve conduction block in one leg by continuoussuperfusion with TTX or only continuous, systemic infusion of TTX; both procedures after theinitial reinnervation and thirdly, the acute experiment when the sizes of the motor units and theamount of dual and single innervation in each muscle were determined.

Denervation and reinnervationThe experimental procedure is diagrammed in Fig. 1 A. The muscle nerve of the fourth deep

lumbrical muscle arises from two separate peripheral nerves: the lateral plantar nerve (l.p.n.) andthe sural nerve (s.n.). Branches of these two nerves join at the ankle forming the common l.p.n.This nerve in turn provides branches to a number of foot muscles including the lumbricals.The muscle nerve was exposed by a lateral incision on the plantar surface of the foot, beside the

fifth toe. The nerve was crushed about 2 mm from its entry into the muscle with watchmaker'sforceps, which met at their tips over a distance of at least 3 mm, for about 30 s. The operation wasperformed bilaterally. The wounds were closed with 6-0 silk suture and the animals were allowedto recover. Following this procedure, the reinnervation of the muscle started about 5 days afterdenervation, when the first visible signs of twitching to nerve stimulation could be seen.

Total nerve block and systemic infusion of TTXTen days after denervating the lumbrical muscle, a time when most muscle fibres have become

reinnervated (Fig. 1 B), the right sciatic nerve was prepared for nerve block in one group of rats.The procedure was essentially as described by others (Betz et al. 1980) with a few modifications.Briefy, osmotic mini-pumps (Alzet Model 2002) were filled with 0-9% sterile saline containing TTX(Sigma, 500Wug/ml) and ampicillin (Astra, 200 ,ug/ml). The ampicillin was added to preventbacterial growth in the mini-pump system. An abdominal incision was made and the mini-pumpswere implanted in the peritoneal cavity. Sterile silicone rubber tubing was threaded under the skinof the animal, one end attached to the mini-pump and the other end to a sterile silicone rubbercuff. The cuff (i.d. 3 mm) was fitted loosely around the entire sciatic nerve in the mid-thigh regionsuch that all axons to the hind-foot muscles in the right leg could be blocked (Fig. 1 A). The cuffheld the opening of the tube next to the nerve. The wounds were closed and then the animals wereallowed to recover.Using this procedure it was possible to block nerve impulse conduction in the sciatic nerve

continuously for up to 15 days. Animals which received local superfusion ofTTX to the right sciaticnerve, showed total paralysis of the leg and a complete lack of response to pinching of the righttoe pads from the day after the mini-pump implantation to the day of the acute experiment. Thenerve block was checked daily in all animals with paralysis.

In some animals, the nerve block wore off after a few days. Subsequent post-mortem examinationshowed that the most common explanation was that the cuff had come free of the nerve, or theproximal end of the tubing had become detached from the mini-pump. Occasionally, the distaltip of the tubing had become blocked with a plug of connective tissue.Some animals received systemic TTX infusions only from the tenth day after the bilateral nerve

crush. In these animals osmotic mini-pumps were implanted subcutaneously in the neck region.The pumps contained the same concentration of TTX and antibiotic as the pumps in the previousgroup. Another group of rats had mini-pumps implanted, containing only saline and the antibiotic.

Acute experimentsThe acute experiments were carried out 4-25 days after the bilateral nerve crush. In most animals

with local TTX superfusion of the right sciatic nerve, complete nerve block was still evident at

177

178 T. TAXT

the time of the acute experiment. The rest of the animals with TTX superfusion of the right sciaticnerve showed a partial or complete recovery of sensitivity to pinching the middle toe pads at thattime. No animal with paralysis was allowed to continue for more than 1 day after relief of the nerveblock was first detected.

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Fig. 1. A, Surgical procedure. The muscle nerve to the fourth deep lumbrical muscle wascrushed bilaterally with fine watch-makers forceps. The wounds were closed with one ortwo silk sutures. Ten days later the rat was re-anaesthetized and the right sciatic nerveexposed in the thigh. An osmotic mini-pump filled with TTX (or the solutes only) wasimplanted intraperitoneally and connected to a silastic tube thread subcutaneously fromthe skin opening in the thigh. Here the tube was fixed inside a silastic cuff, which wascarefully placed around the sciatic nerve as indicated in the Figure. All wounds were closedwith silk sutures. After each surgical procedure each rat received 30,000 i.u. procainepenicillin intramuscularly. M.p.n., medial plantar nerve. B, reinnervation curve forcontrol muscles with no TTX infusion. The extent of reinnervation is given as the ratiobetween the maximum nerve-evoked twitch tension and the maximum direct twitchtension. The regenerating motor axons started to form functional synapses in the muscle4-6 days after the muscle nerve crush. The reinnervation was virtually complete 10 daysafter the crush. Twitch and tetanic tension measurements gave similar results.

Animals with complete anaesthesia of the toe pads were first anaesthetized with ether and theright sciatic nerve was exposed in the leg and cut proximal to the cuff to avoid reflex contractionsof thigh muscles. The sciatic nerve was then stimulated with a pair of silver wires, using 10 V, 0-1 mspulses. Stimulation above or within the cuff never caused any contraction of muscles in the leg,while stimulation below the cuffalways caused a vigorous contraction of the foot. This test thereforeconfirmed the assessment of the nerve block from the daily test of local anaesthesia.The animals were killed and the lumbrical muscles on both sides were dissected with the distal

tendon inserting on the fifth toe and the l.p.n. and the s.n. intact. The fifth toe was cut apart fromthe foot at the proximal interphalangeal joint. The mini-pumps from animals with systemic infusiononly were cut in two pieces to check that the pump had been partly or completely emptied duringthe infusion period.The muscles were mounted in a Sylgard lined chamber and superfused continuously with Liley's

solution (Liley, 1956) gassed with 95% 02/5% CO2. For tension measurements the fifth toe wasmounted in the bath with small stainless-steel pins to fix the distal tendon of the muscle. The

EFFECTS OF TTX ON SYNAPSES

proximal tendon was attached to a strain gauge (Aker Engineering, 15-3 mV/N) with silk threadand the muscle was stretched to the approximate resting length. The angle between the muscleand the fifth toe was approximately 900. The muscle length was adjusted to produce the maximumnerve-evoked twitch tension. No further length adjustments were done during the tensionmeasurements. The maximum nerve-evoked twitch never decreased more than 10% of the initialvalue during all the tension measurements.The s.n. and the l.p.n. were stimulated separately with 1-10 mV, 0-2 ms pulses of both polarities

and it was always checked that cross-stimulation did not occur. The maximum twitch tensionsproduced in the muscle by supramaximal stimulation of each nerve alone and by both nervestogether were recorded. Then the maximum nerve-evoked twitch tension was compared to themaximum direct twitch tension of the muscle. Maximum direct tensions were obtained by placinga pair of silver wires close to opposite sides of the muscle and by sending 50 V, 1 0 ms pulses betweenthem. Subsequently, the nerve stimulus to each of the nerves was carefully graded to record allthe individual motor-unit tensions. Both polarities were used and each motor unit was observedseveral times. The storage capability of the oscilloscope greatly facilitated these measurements.Following the twitch-tension measurements the corresponding tetanic tensions (50 Hz) wererecorded, except for the individual motor-unit tensions. There was no increase in the maximumtetanic tension of the muscles with stimulation frequencies higher than 50 Hz.To obtain as accurate estimates of the extent of polyneuronal innervation as possible, all

calculations involving polyneuronal innervation were based on the s.n. motor-unit sizes. The s.n.supplies usually only one to three motor units to the muscle, while the l.p.n. supplies eight to twelve(Betz, Caldwell & Ribehester, 1979). Hence, by this procedure most errors due to overlap betweendifferent motor units and errors due to non-linear summations of tensions were avoided.The maximum nerve-evoked and direct tetanic tensions increased a few per cent during the first

few trains of stimulation (50 Hz for 08-10 s, with at least 30 s interval). The calculations of thetetanic-tension overlap (Brown & Matthews, 1960b) were therefore based on two sets of tetanic-tension values for each muscle, measured when the maximum nerve-evoked and direct tetanictensions were stable. The first set of measurements was obtained by stimulating s.n. alone, l.p.n.alone and then both nerves together, while the second was obtained by stimulating both nervestogether, s.n. alone and then l.p.n. alone. The mean of these two tension overlap measurementswas calculated.From each set of the tetanic-tension overlap measurement the apparent polyneuronal innervation

of the s.n.-innervated fibres was calculated; s.n. (% poly) = 100% x (L+S-LS)/S. Here L is thetetanic tension the muscle produces by supramaximal stimulation (50 Hz) of the lateral plantarnerve alone, S is the tetanic tension produced by supramaximal stimulation of the s.n. alone andLS is the maximum tetanic tension produced by supramaximal stimulation of both nervessimultaneously. The average size of the s.n. motor units was calculated from the tension obtainedon tetanic stimulation, the number of s.n. motor units and the maximum tetanic muscle tension;s.n. (motor-unit size, %) = (S x 100)/(Ns x Max. tetanic tension). Ns is the number of s.n. motorunits. This method was used to avoid errors introduced by non-linear summation of twitch responsesdue to imperfect isometric recording conditions (Brown & Matthews, 1960b).

It was also of interest to calculate the fraction of the muscle which received suprathreshold inputonly from s.n. When both nerves were stimulated together (50 Hz), the tension obtained was greaterthan when l.p.n. alone was stimulated. The difference in tension must therefore be due to fibreswith suprathreshold inputs only from s.n. This difference, expressed as a percentage of the totaltension, was shared among the number of s.n. motor units counted by twitch-tension measurements;s.n. only (% total tension) = ((1 -LILS) x 100)/N8.The amount of dual and single innervation of muscle fibres was also determined in most muscles

by making intracellular recordings from cut-muscle-fibre preparations (Barstad, 1962) with glassmicropipettes filled with 5 M-potassium acetate (10-20 MCI). The muscle was stretched flat in thebath after that the proximal and distal quarter of the muscle had been cut away with a small pairof scissors. By using a dark-field condenser all the terminal branches of the motoneurones were easilyseen in these flat muscles. Intracellular recordings were therefore only done close to these branches.Each muscle was surveyed systematically. The number of s.n. innervated fibres recorded from wasat least ten in each muscle, and usually it was larger than twenty.Zinc iodide-osmium staining (Akert & Sandri, 1968), following the procedure described in Betz

et al. (1980), was done in some muscles to study the synaptic morphology of normal and reinnervated

179

lumbrical muscle fibres. Also a few muscles were stained for acetylcholinesterase (Buckley & Heaton,1968) to investigate the distribution of end-plates in whole muscles. The Student's t test was usedto compare mean values, while a modified X-square test (Homogeneity test, Sverdrup, 1964) wasused to compare the two distributions. All values are given as mean+ S.D. (n).

RESULTS

The results are organized in three parts. First, the extent of polyneuronalinnervation in normal lumbrical muscle is described and compared to the extent ofpolyneuronal innervation in reinnervated muscles at different time intervals after thedenervation. The reinnervated muscles were of four kinds. The first group was fromanimals with no TTX infusion. The next group was from animals with their legsparalysed for 5-15 days by local superfusion of the sciatic nerve with TTX. The thirdgroup consisted of muscles from legs contralateral to the paralysed legs, while thelast group was from animals receiving only TTX systemically.

Secondly, the number and sizes of motor units in normal and in the four kinds ofreinnervated muscles are presented. Finally, follows a description of a few contractileproperties of these muscles.

Polyneuronal innervation of normal and reinnervated musclesThe extent of polyneuronal innervation was estimated by tetanic-tension overlap

measurements and intracellular recordings. The tetanic-tension measurements gavean estimate of the fraction of the muscle fibres innervated by suprathreshold inputsfrom both the s.n. and l.p.n., while the intracellular recordings gave a reasonableexpression for the total extent of polyneuronal innervation by all sub-threshold andsuprathreshold inputs.

Normal lumbrical muscleTension measurements. Normal, adult skeletal mammalian muscle fibres are usually

innervated only by single motor axons, and isometric tetanic contractions of motorunits summate virtually linearly (Brown & Matthews, 1960b; Baker & Ip, 1966;Tuffery, 1971). In order to obtain a base-line value for comparison with reinnervatedmuscles the tetanic-tension overlap was re-examined in the normal lumbrical muscle.The average degree ofapparent polyneuronal innervation by tetanic-tension measure-ments was 64+ 36 % (7), which is slightly higher than reported in other normalmuscles (Brown & Matthews, 1960b), Figs. 2A and 3A.

Intracellular recordings. The increased extent ofpolyneuronal innervation observedby tension measurements could be due to mechanical factors (Brown & Matthews,1960b) or to true polyneuronal innervation. During synapse elimination in normaland reinnervated muscles some of the e.p.p.s. may pass through a stage in which theyare sub-threshold (Dennis & Yip, 1978; Taxt, Ding & Jansen, 1983). Estimates ofthe polyneuronal innervation by tension measurements in such situations willtherefore underestimate the true extent of polyneuronal innervation. Hence, it wasimportant to check the results based on tension measurements with intracellularrecordings.

Unexpectedly, about 25% ofthe s.n.-innervated muscle fibres were also innervatedby l.p.n. on intracellular recording (Fig. 5A). Usually one of the inputs was much

180 T. TAXT

EFFECTS OF TTX ON SYNAPSES

smaller than the other. In more than 60% of thirty-one dually innervated fibres fromseven muscles the size of one of the end-plate potentials (e.p.p.s) was less than 10%of the size of the other e.p.p. in the same fibre (Fig. 4A) and often no larger thanthe miniature end-plate potentials (m.e.p.p.s). These small e.p.p.s were thereforealmost certainly sub-threshold. Only eight out of the thirty-one dually innervatedfibres had a small e.p.p., which was more than 30% of the size of the larger e.p.p.in the same fibre and possibly suprathreshold (Fig. 4B).

A B C

11mN 13mN 11mN

200 ms 200 ms 200 ms

D E F

511mN N -llmN 11 mN

200 ms 200 ms 500 ms

Fig. 2. Polyneuronal innervation demonstrated by summation of tetani. Maximum s.n.tetanic tension: lower trace; maximum l.p.n. tetanic tension: middle trace; maximumnerve-evoked tetanic tension by simultaneous stimulation of both nerves: upper trace. A,normal muscle; B, reinnervated muscle from an animal not receiving TTX, 14 days afterthe muscle nerve crush; C, reinnervated muscle 23 days after the muscle nerve crush andparalysed for 12 days; D, reinnervated muscle 23 days after the muscle nerve crush,contralateral to C; E, reinnervated muscle 19 days after muscle nerve crush from an animalwith only systemic infusion of TTX. F, maximum direct and indirect tetanic tensions ofa reinnervated muscle 21 days after the muscle nerve crush and paralysed for 11 days.This record illustrates the after-contraction following both the direct and indirect tetanicstimulations in the paralysed muscles after the stimulus has been turned off. Notedifferences in time scale. The estimated frequencies of polyneuronal innervation based ontetanic-tension overlap in these muscles were: A, 6-8 %; B, 38-1 %; C, 78-6 %; D, 38-5 %;E, 32-3%.

The synapses of these dually innervated fibres were close to each other since therise times of the two e.p.p.s in the same fibre were equal in most cases. To illustratethe difference in sizes of the e.p.p.s in the polyneuronally innervated fibres a diagramshowing the ratios between the small and the large e.p.p.s was constructed (Fig. 6A).

In dually innervated fibres, the latency of the large e.p.p.s of the s.n. motor axonswas 2-8 + 0'5 ms (10), while that of the small e.p.p.s was 2-9 + 0-5 ms (24). Similarly,the latency of the large e.p.p.s of the l.p.n. motor axons was 3-2 + 0-5 ms (24) andthat of the small e.p.p.s 3-6+0-7 ms (10). The latency of the small e.p.p.s was notsignificantly longer than the latency of the large e.p.p.s in any of these two cases(P>0-1).

Staining of the nerve terminals with zinc iodide-osmium provided additionalsupport for the existence ofpolyneuronal innervation in normal lumbrical muscle, but

181

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Fig. 3. The percentage polyneuronal innervation of s.n. innervated fibres in the lumbricalmuscle estimated by the tetanic-tension overlap technique as a function of days afterbilateral muscle nerve crush. A, I, normal lumbrical muscle; E, reinnervated controlmuscle with no additional treatment; *, reinnervated control muscle with systemicinfusion of ampicillin in saline. B, *, reinnervated muscle paralysed with TTX superfusionon the nerve; 0. reinnervated control muscle contralateral to paralysed muscle; x,reinnervated muscle with systemic infusion ofTTX and ampicillin in saline. The data aredivided in random intervals of 5 days. P < 001 (one-sided) comparing reinnervatedcontrol muscles denervated more than 25 days earlier and normal muscles. P> 0.10(one-sided) comparing reinnervated control muscles denervated 21-25 days earlier withand without ampicillin infusion. P < 0005 (one-sided) comparing reinnervated paralysedmuscles and reinnervated contralateral control muscles denervated 21-25 days earlier.

did not indicate whether the majority of the polyneuronal innervation was due tomaintenance of the neonatal state or due to sprouting and regression of axon.terminals as seen in normal, adult frogs (Wernig, Pecot-Dechavassine &; Stover, 1980).In four muscles all fibres studied had only one mature end-plate. In addition, a fewof the muscle fibres were innervated close to the end-plate by small non-myelinatedterminal sprouts from a neighbouring fibre. The end-plate of some others receivedinnervation from two myelinated axons which could not be seen to originate froma common axon (see Barker &r Ip, 1966; Tuffery, 1971). The frequency of suchobservations were small (1*5 %/ of each), but must be considered only as a lower limitbecause only unequivocal cases were included. These histological investigationsdemonstrated that some muscle fibres were polyneuronally innervated and that thesynapses of the dually innervated fibres were located at closely neighbouring sites.Furthermore, fine nerve fibres were seen microscopically only in the middle part ofthe muscle where the e.p.p.s were recorded and there was only one sharply definedend-plate band by staining the end-plates for acetylcholinesterase in two muscles.

182 T. TAXT

EFFECTS OF TTX ON SYNAPSES

Reinnervated muscles with no TTX infusionMammalian skeletal muscle is transiently polyneuronally innervated during re-

innervation following a muscle nerve crush (McArdle, 1975). To provide referencevalues for the time course of this polyneuronal innervation in the lumbrical muscle,reinnervated muscles were examined at various time intervals after the muscle nervecrush.

Tension measurements. The maximum average extent of polyneuronal innervationestimated by tetanic-tension deficit was reached about 13-15 days after the musclenerve crush (Figs. 2A and 3A), and was slightly larger than the maximum extent

A B C

D E F

CX t ~~~~~ 8 < <~~~4mVL.p.n. S.n. L.p.n. S.n. L.p.n. S.n. 10 ms

Fig. 4. Intracellular recordings from normal and reinnervated lumbrical muscle. A, normalmuscle fibre receiving one tiny and one large e.p.p.; B, normal fibre receiving one largeand one rather small e.p.p., a m.e.p.p. is also visible on this record; C, reinnervated fibrefrom an animal with infusion of only ampicillin in saline receiving one rather small andone large e.p.p. D, reinnervated, paralysed fibre receiving two large and one small e.p.p.;E, reinnervated, paralysed fibre receiving two large e.p.p.s with slightly different timecourses; F, reinnervated, paralysed fibre receiving one presynaptic axon which is firingtwice after a single stimulus; G, H, reinnervated fibres from muscle contralateral toparalysed muscle. The fibre in a receives two large e.p.p.s with variable sizes, while thefibre in H receives one large and one small e.p.p.; I, reinnervated fibre from an animalwith only systemic TTX infusion and receiving two rather large e.p.p.s. The survival timeafter the bilateral nerve crush was for C, 66 days; D, 21 days; E, 20 days; F, 21 days;0, 21 days; I, 25 days. s.n.: sural nerve, l.p.n.: lateral plantar nerve.

of polyneuronal innervation in the extensor digitorum longus muscle during re-innervation estimated by intracellular recordings (McArdle, 1975). The extent ofpolyneuronal innervation decreased subsequently, but remained higher than innormal muscles even more than 4 weeks after the denervation (Fig. 3A).

Intracellular recordings. By intracellular recording the maximum extent of

183

polyneuronal innervation was about 50 %. It was reached 10-15 days after dener-vation, which was similar to that observed by tetanic-tension measurements. On theother hand, by 16-20 days after denervation there was no significant differencebetween the extent of polyneuronal innervation of reinnervated and normal musclesbased on intracellular recordings. The apparent discrepancy in the estimates ofpolyinnervation obtained by tension measurements and intracellular recordings(Figs. 3A and 5A), may be explained by the different ratios of the small and largee.p.p.s ofdually innervated fibres in the reinnervated and normal muscles (Fig. 6 A-C).

Muscles paralysed by TTX superfusion of the sciatic nerveThese muscles were paralysed for 5-15 days from the tenth day after the muscle

nerve crush. The nerve block started therefore at a time when virtually all musclefibres already received strong synaptic input from the reinnervating motor axons (seeFig. 1B).

Tension measurements. After a week of complete paralysis the TTX block causedan increase up to 80% in the extent of polyneuronal-innervation of the muscle fibresby the reinnervating motoneurones (Fig. 2 C). Subsequently, there was no sign of anynet elimination of this polyneuronal innervation during blocking periods lasting upto 15 days (Fig. 3B).

Intracellular recordings. There was a close correspondence between the extent ofpolyneuronal innervation measured by intracellular recordings and tension measure-ments, indicating that most of the polyneuronally innervated fibres received at leasttwo independent, suprathreshold inputs (Figs. 3B and 5B).

Indeed, the e.p.p.s of the polyneuronally innervated fibres were in most cases bothlarge and three distinct e.p.p.s were seen in many muscle fibres (Fig. 4D).Correspondingly, the diagram of the ratios between the small and the large e.p.p.sin the polyneuronally innervated fibres for these muscles showed a much largerfrequency of e.p.p.s with approximately the same size than that of comparablereinnervated control muscles (Fig. 6C, D).

In some dually innervated fibres of muscles paralysed for more than 9 days, bothe.p.p.s were large, and yet the time course of one of the e.p.p.s was clearly slowerthan that ofthe other (Fig. 4E). Histologically, there was no indication of innervationoutside the original end-plate region in these muscles. Fine nerve fibres were seenmicroscopically only in the middle part of the muscle where the e.p.p.s were recorded.Furthermore, no more than one end-plate was seen on fibres where the end-plateswere stained with zinc iodide-osmium and the muscle teased in small, fibre bundles.Fine sprouts from end-plates on neighbouring muscle fibres reached several of theend-plates in the paralysed muscles. The slow e.p.p.s could therefore be due to a lowlevel of acetylcholinesterase at the parts of the end-plates formed and innervatedduring the paralysis (Koenig & Vigny, 1979; Cangiano, L0mo, Lutzemberger & Sveen,1980).

Muscles contralateral to the paralysed musclesTension measurements. These muscles were initially investigated to obtain paired

controls to the paralysed muscles. The maximum degree of polyneuronal innervationreached about 50% which was not significantly different from that of reinnervated

184 T. TAXT

EFFECTS OF TTX ON SYNAPSES 185

muscles in animals which did not receive any TTX (Figs. 2D and 3A, B).Unexpectedly, however, the high degree of overlap remained essentially unchangedas long as the rats received TTX, in contrast to the normalization which took placein reinnervated muscles of animals with no TTX administration. Therefore, at 15-20days and at 21-25 days after denervation the extent ofpolyneuronal innervation wassignificantly larger P < 0-025) in contralateral control muscles than in controlmuscles receiving no TTX.

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Normal 11-15 16-20 21-25 26-30 65-125 11-15 16-20 21-25Days after nerve crush

Fig. 5. Per cent polyneuronal innervation of s.n. innervated fibres in lumbrical muscleestimated by intracellular recording as a function of days after bilateral muscle nervecrush. Symbols as in Fig. 3. P < 0-01 (one-sided) comparing reinnervated paralysedmuscles and reinnervated contralateral control muscles 21-25 days after the denervation.

Intracellular recording. Initially, at 14-17 days after the muscle nerve crush, theextent of polyneuronal innervation measured by tension techniques was notsignificantly different from that estimated by intracellular recordings. In the periodof21-25 days after denervation, however, the degree of polyneuronal innervation wassignificantly larger by intracellular recording than by tetanic-tension measurements(Figs. 3B and 5B). The divergence between the estimates ofthe extent ofpolyneuronalinnervation by the two techniques was again probably caused by there being a largefraction of dually innervated fibres with one small, sub-threshold e.p.p. and one largee.p.p. (Fig. 4G, H). The diagram of the ratios between the small e.p.p. and the largee.p.p. in the dually innervated fibres for these muscles at 21-25 days after the nervecrush was different from that of normal and paralysed muscles. (Fig. 6A, D, E). Asexpected from the tetanic-tension measurements, it also differed from the nerve-dominance diagram for reinnervated muscles of animals with only bilateral nervecrush and no TTX infusion (Fig. 60, E).

186 T. TAXT

A B C100-

n= 7 n= 5 n=8

80 m= 125 m=79 m=110

>. 60UC

40-

20 -

0-1 2 3 4 1 2 3 4 1 2 3 4Dual - s.n. Dual -+ s.n. Dual -+ s.n.

D E F100

n =6 n =7 n = 3

80 m=74 m= 115 m=55

60C

0f40,L20-0 J Ij i1

1 2 3 4 1 2 3 4 1 2 3 4Dual -+ s.n. Dual -* s.n. Dual -+ s.n.

Fig. 6. The ratio between the small and the large e.p.p. in muscle fibres innervated bothby l.p.n. and s.n. and the fraction of s.n. innervated fibres also innervated by l.p.n. Alldiagrams are frequency distributions and a 100% is all s.n. innervated fibres of thatparticular kind of muscle. Group 4 is muscle fibres with an input only from s.n. Duallyinnervated fibres in which the e.p.p. to l.p.n. stimulation was between one-half and twotimes the size of the responses to s.n. stimulation were placed in group 2. Fibres in whichthe l.p.n. responses was more than twice as big as the s.n. response were placed in group1. Fibres where the s.n. response was more than twice the size of the l.p.n. response wereplaced in group 3. n, number of muscles; m, number of s.n.-innervated fibres recordedfrom. A, normal muscle; B, reinnervated control muscle 11-15 days after denervation withno additional treatment; significantly different from the diagram ofnormals (P < 0-0005).C, reinnervated control muscle 21-25 days after denervation with no additional treatmentor with systemic infusion of ampicillin in saline; significantly different from the diagramofnormals P < 0 05). D, reinnervated muscle 21-25 days after denervation paralysed withTTX; significantly different from the diagram ofcontralateral control muscles (P < 0 025),and that of reinnervated control muscles with no additional treatment or with systemicinfusion of ampicillin in saline (P < 0 0005). E, reinnervated control muscle contralateralto paralysed muscle 21-25 days after denervation; significantly different from the diagramof normals (P < 00005) and that of reinnervated control muscles with no additionaltreatment or with systemic infusion of ampicillin in saline (P < 0-0005). F, reinnervatedmuscles 21-25 days after denervation with systemic infusion of TTX and ampicillin insaline; not significantly different from the diagram of contralateral control muscles(P > 0-10).

EFFECTS OF TTX ON SYNAPSES

Muscles from animals with only systemic TTX infusionThe maintenance of the high extent of polyneuronal innervation in muscles

contralateral to paralysed muscles might have been due to a sprouting stimulusproduced by the paralysed muscles (Thompson et al. 1979), or by the blockedmotoneurones as in the case of the central sprouting signal from contralateralaxotomized motoneurones reported in frogs (Rotschenker, 1979). Alternatively, itcould be due to a systemic effect of the TTX on the muscle fibres or the motoneurones.To distinguish between these possibilities TTX was administered systemically to aseparate batch of rats.

Tensionmeasurements. The extent ofpolyneuronal innervation ofmuscles from theseanimals reached the same maximum values as the polyneuronal innervation ofmuscles contralateral to the paralysed muscles (Figs. 2E and 3B). These effects onthe polyneuronal innervation were not due to the mini-pump itself or the ampicillinin the TTX solution because the maximum extent of polyneuronal innervation andits time course in muscles from animals with osmotic mini-pumps containing onlyampicillin in saline were similar to those of reinnervated muscles from animals withno pump implants (Fig. 3 A).

Intracellular recording. One small and one large e.p.p. were seen in most ofthe duallyinnervated muscle fibres, while a few fibres received two approximately equal, largee.p.p.s (Fig. 4 I). The time course and degree of the polyneuronal innervation were asin muscles contralateral to paralysed muscles (Figs. 3B and 5B). The diagrams ofthe ratios between the small and the large e.p.p.s in dually innervated fibres of thesetwo types of TTX-exposed muscles did not differ significantly (Fig. 6E, F).

Numbers and sizes of the motor units in normal and reinnervated musclesThe implantation ofa cuff around the sciatic nerve could by accident cause a partial

denervation of the lumbrical muscle on the operated side, for instance by pressureon the nerve. Partial denervation gives rise to sprouting ofthe remaining motoneurones(Brown & Ironton, 1978; Thompson, 1978), and could therefore possibly be theexplanation for the increased extent of polyneuronal innervation in the paralysedmuscles. However, there was no significant difference in the numbers of motor unitsin normal muscles, reinnervated muscles from animals with no TTX infusion,paralysed muscles, contralateral control muscles and muscles from animals with onlysystemic TTX infusion (Table 1). Also, the total number of muscle fibres in theparalysed muscles, 927 + 9 (3), was not significantly different from that ofcontralateralcontrol muscles, 927 + 81 (6). The differences in the extent ofpolyneuronal innervationbetween the various kinds of muscles cannot therefore be explained by differentinnervation ratios (Bixby & Van Essen, 1979) or by accidental partial denervations.Since the number of motor units in all the four kinds of reinnervated muscles wasnormal, it was expected that the average s.n. motor-unit tensions (i.e. motor-unitsizes) of these muscles would be larger than in normal muscles as long as the extentofpolyneuronal innervation measured by tension overlap was increased. This was alsothe case (Fig. 7). However, the average s.n. motor-unit size was only 1-3 times as largein the paralysed muscles as in the contralateral control muscles (Table 1). On the otherhand, the average fraction of each paralysed muscle innervated by one s.n.

187

188 T. TAXT

motoneurone and no l.p.n. motoneurones was decreased to 2-6 + 1'5% (7) 21-25 daysafter the nerve crush, while it was 6-1 + 3-1 % (6) for contralateral control muscles and7 3 + 3 1 % (9) for reinnervated muscles from animals with no TTX administration.The fraction for normal muscles was 60+ 1-3% (7). (See R. R. Ribehester & T. Taxt,in preparation, for the significance of these points.)

A B30-

C0

C

+120-

o 0 X

04 0SN 0

2°°° 2 * 0C 0O

S 0 0

o 10 o~

o ~~~~~~3 08

c/i 0~~~~~~~

0 // I ~ ~~ ~~~~~I I/Normals 11-15 16-20 21-25 26-30 65-125 11-15 16-20 21-25

Days after nerve crush

Fig. 7. Mean s.n. motor unit sizes in the lumbrical muscle estimated by twitch andtetanic-tension measurements as a function of days after bilateral muscle nerve crush.Symbols as in Fig. 3. Each point represents the mean s.n. motor-unit size in per cent ofthe maximum direct tetanic tension from one muscle (see Methods for calculations). 16-20and 21-25 days after the muscle nerve crush the mean sizes for the paralysed muscles,the contralateral control muscles and for the muscles from the animals with systemic TTXinfusion was significantly larger (P < 0-025) than the mean size for normal lumbricalmuscles.

Contractile Properties of normal and reinnervated musclesIn this part of the study the maximum (direct or indirect) tetanic tension, the

tetanus/twitch ratio and the rise time of normal and reinnervated muscles wereinvestigated to further characterize the effects of local and systemic TTX infusionon the lumbrical muscle.

There was no significant reduction in the maximum tetanic tension of musclesparalysed for up to 15 days compared with contralateral control muscles (Table 1).On the other hand, the tetanic-tension recordings of muscles paralysed more than10 days regularly showed a large after-contraction lasting several hundred millisecondsafter the stimulus had been turned off (Fig. 2F). Tetanic-tension records fromcontralateral control muscles and from muscles of animals with only systemic TTXinfusion showed a much smaller after-contraction in a few cases and usually no suchafter-contraction 20 days or more after the denervation. Muscles from animals whichdid not receive any TTX did not produce any significant after-contraction followinga tetanic stimulation except very early after reinnervation.The direct tetanus/twitch ratio of the paralysed muscles was reduced compared

EFFECTS OF TTX ON SYNAPSES 189

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to that of contralateral control muscles and normal muscles, while the rise time ofthe twitch was clearly increased (Table 1). The reduction in the tetanus/twitch ratiowas largely due to increased maximum nerve-evoked and direct twitch tensionbecause the maximum tetanic tension remained virtually unchanged compared tothat of contralateral control muscles.

It is likely that most of the increase in the twitch tension and at least part of theincrease in the twitch rise time were caused by double firing of the blocked motoraxons. Two consecutive, similar e.p.p.s were seen on intracellular recording in severalparalysed muscle fibres after a single nerve shock lasting 0-2 ms and just strongenough to excite the axon, Fig. 4F (see also R. R. Ribehester & T. Taxt, inpreparation). The last of two such consecutive e.p.p.s often disappeared after a fewstimuli at 0-5 Hz without any change in the stimulus parameters or the size of thefirst e.p.p. The two consecutive e.p.p.s were usually larger and had longer decay timesthan e.p.p.s in unparalysed muscles fibres.

Denervation ofmuscles gives rise to a reduced concentration ofthe 16 S form ofacetylcholinesteraseat the neuromuscular junction (Hall, 1973). Also, activity of the muscle is necessary to restore this16 S form of acetylcholinesterase at the end-plate (Koenig & Vigny, 1979; Weinberg & Hall, 1979;Cangiano et al. 1980). Therefore, it is possible that the double firing in the paralysed muscles was

due to local accumulation of acetylcholine caused by a low level of the 16 S form of acetyl-cholinesterase at the previously denervated neuromuscular junctions.

This is consistent with results showing that repetitive 'back-firing' in mammalian nerve-musclepreparations occurs at a very small percentage of end-plates normally, but is enhanced bycholinesterase inhibitors (Masland & Wigton, 1940; Barstad, 1962; Randic & Straughan, 1964).Presumably, this effect is caused by a reduced concentration of functional aectylcholinesterase atthe end-plates. In addition, acetylcholinesterase inhibitors are known to produce repetitive'back-firing' in the muscle nerve after tetanic contractions (Masland & Wigton, 1940). A low levelof acetylcholinesterase at the end-plates of paralysed muscles could therefore also explain the largeafter-contractions in these muscles.

Irrespective of the cause ofthe double firing, it is unlikely that activity in one motorunit caused activity in other motor units during the intracellular recordings andduring the twitch and tetanic contractions of the paralysed muscles, for instance byephaptic stimulation of motor axons (Brown & Matthews, 1960a). First, there was

not a 100 % overlap between the s.n. andl.p.n. contractions in any of the five cases

where the s.n. contained only one motor axon which innervated the paralysed muscle(see, for example, Fig. 20). Secondly, the tetanus/twitch ratios of the s.n. andl.p.n.contractions from the paralysed lumbrical muscles which received more than one s.n.

motor axon were not significantly different from the corresponding ratios of paralysedmuscles which received only one s.n. motor axon (P> 010). Hence, it is also likelythat in these latter cases there was no spread of activity from one motor unit toanother. Thirdly, the mean latency of the last of the two consecutive e.p.p.s observedin eight paralysed muscle fibres was 13-8 +3-8 ms (range 8-17ms), and the mean

latency of the e.p.p. of thirty-one singly innervated paralysed fibres was 4-8 + 0-6 ms(range 4-7ms). Because of this difference in latencies it is unlikely that the e.p.p.s

of singly innervated paralysed fibres and the last of the two consecutive e.p.p.s inother paralysed fibres were directly related to each other.There was no differences in the contractile properties mentioned above when

contralateral control muscles, muscles from animals with only systemic TTX infusion,or control muscles from animals receiving no TTX were compared (Table 1).

190 T. TAXT

EFFECTS OF TTX ON SYNAPSES

DISCUSSION

Polyneuronal innervation of normal muscle. It was unexpected to find as many as25% ofthe adult lumbrical muscle fibres polyneuronally innervated. The small e.p.p.sseen in almost all the dually innervated fibres were probably not caused by electricalsynapses between neighbouring muscle fibres as seen in embryonic mammalianskeletal muscles (Dennis, Ziskind-Conhaim & Harris, 1981), because the rise timesof the e.p.p.s in the same fibre were equal in most cases. Furthermore, there is noevidence for electrical synapses between rat muscle fibres more than one week afterbirth (Brown et al. 1976; Schmalbruch, 1982). In addition, small e.p.p.s of s.n. motoraxons in dually innervated muscle fibres were found even in two muscles where thesingle s.n. motor axon which innervated the muscle was stimulated separately. Sinceeach motoneurone normally innervates fibres widely dispersed through the cross-section of the muscle (Kugelberg, 1973), it is unlikely that these small e.p.p.s couldbe caused by electrical synapses from neighbouring fibres receiving large e.p.p.s fromthe s.n. The latter observation also makes unlikely that ephaptic stimulation of asmall region of a nerve terminal as the result of a large orthodromic electrical fieldcaused by synchronous stimulation of a large fraction of the neighbouring musclefibres could explain the small e.p.p.s.

Since one of the e.p.p.s of almost all dually innervated fibres was very small whilethe other e.p.p. was large, it is probable that the small e.p.p. was sub-threshold evenat normal membrane potentials. This explains the difference in the extent ofpolyneuronal innervation of normal lumbrical muscle found by tetanic-tensionmeasurements and by intracellular recording in cut-muscle-fibre preparations.

It is reasonable to ask why this level of polyneuronal innervation has not been seenbefore. The procedure for measuring the extent of polyneuronal innervation byintracellular recording in the present experiments differed in three important respectsfrom previous investigations concerning polyneuronal innervation of mammalianskeletal muscle (Redfern, 1970; McArdle, 1975; Brown et at. 1976). First, a cut-muscle-fibre preparation was used such that m.e.p.p.s of 0-3-2-0 mV size wereregularly seen at the same time that the e.p.p.s in the same fibre were recorded.Therefore, the resolution was good and allowed detection of evoked e.p.p.s as smallas spontaneous m.e.p.p.s in many of these muscle fibres.

Secondly, the motor axons of the lumbrical muscle were stimulated through twodifferent peripheral nerves which allowed time separation of e.p.p.s coming from themotor axons in the two nerves. Thereby it was possible to detect a tiny e.p.p. froma motor axon in one nerve which had a higher firing threshold than an axon in theother nerve which produced a large e.p.p. in the fibre.

Thirdly, the extent of polyneuronal innervation was estimated as the fraction of-the muscle fibres innervated by the s.n. which also was innervated by the l.p.n.Because the s.n. in most cases contained only one to three of the ten to fourteenlumbrical motoneurones, very few tiny e.p.p.s of the s.n. passed undetected.

In addition, all the present results were obtained from the fourth deep lumbricalmuscle and only from young, adult rats. It is possible that the extent of polyneuronalinnervation is lower in other muscles and decreases in all muscles as the rats growolder.

Betz et at. (1979) used the fourth deep lumbrical muscle to study elimination of

191

polyneuronal innervation in neonatal rats, but failed to observe any significant,persisting polyneuronal innervation. However, from the description of their intra-cellular recording technique, it is not clear whether they had the same resolution intheir recordings as in this study. Furthermore, they did not estimate the extent ofpolyneuronal innervation as the fraction of muscle fibres innervated by the s.n. whichwas also innervated by the l.p.n., and they have only two observations based onintracellular recordings from rats older than 20 days.

Reinnervation ofthe lumbrical muscle after nerve crush only. Calculating from the firstday e.p.p.s could be recorded after the denervation, the time course ofthe polyneuronalinnervation was similar to that ofthe rat extensor digitorum longus muscle (McArdle,1975). In both cases the maximum extent of polyneuronal innervation was reachedabout 9-11 days after the start of the reinnervation. Also, the diagram of the ratiosbetween the small and the large e.p.p.s in dually innervated fibres of the lumbricalmuscles 25 days or more after the denervation was not significantly different fromthat of a normal muscle. At 65 days or more the extent of polyneuronal innervationwas reduced almost to normal levels for both kinds of muscles. This is in contrastto what happens after partial denervations where a level of polyneuronal innervationhigher than normal seems to persist (Brown & Ironton, 1978; Thompson, 1978).

Polyneuronal innervation of paralysed muscles. The extent of polyneuronal inner-vation in these muscles increased to 80-100% of the muscle fibres after a week ofparalysis and there was subsequently no net elimination of synapses. This extent ofpolyneuronal innervation was much larger than that reported for the paralysed soleusmuscle of adult rats (Benoit & Changeux, 1978). Since the high level of polyneuronalinnervation remained stable during paralysis, the net loss of synapses seen inreinnervated muscles appears to require nerve or muscle activity to occur. However,these conclusions are limited to the first three to four weeks of reinnervation.

Systemic effect of TTX. Sprouting and increased extent ofpolyneuronal innervationare seen in contralateral control muscles of frogs after denervation of one cutanouspectoris muscle (Rotschenker, 1979). The sprouting signal is thought to be mediatedtransneuronally within the spinal cord from the axotomized motoneurone pool to thecorresponding contralateral pool of intact motoneurones. However, the higher degreeof polyneuronal innervation of muscles contralateral to the paralysed musclesreported in this paper was not caused by a similar sprouting signal produced by theparalysis (see also Brown, Holland & Ironton, 1980). The effect was mimicked by onlysystemic TTX infusion and bilateral lumbrical nerve crush. Clinically these rats wereapparently normal. In addition, reinnervated muscles from animals with no osmoticpump or from animals with osmotic pumps filled with 0 9% saline and 200 ,ug/mlampicillin showed much less polyneuronal innervation than the two groups ofTTXexposed muscles. It is therefore most likely that the increase extent of polyneuronalinnervation observed in TTX exposed, active muscles was due to a systemic effectof TTX. The mechanism of such a TTX effect is unknown.The systemic effect of TTX prevented the net elimination of synapses that

normally occurred following muscle reinnervation. This could either be brought aboutby equal non-zero rates of formation and elimination of synapses or by virtually nosynapse formation and synapse elimination at all, 15-25 days after the nerve crush.In any case, it is important to interpret with great care the results obtained by

192 T. TAXT

EFFECTS OF TTX ON SYNAPSES

application of TTX in the peripheral and central nervous system to study howimpulse activity influences the formation and elimination of synapses. Particularly,contralateral controls with the same systemic exposure to TTX as the blockedneurones should be used whenever it is possible.

It is a pleasure to thank Dr J. K. S. Jansen for inspiring discussions and support during this studyand Dr R. R. Ribehester for taking part in some of the experiments and also for their helpfulcriticism on earlier drafts of this manuscript. Additional thanks go to HAvard T0nnesen for excellenttechnical assistance, to Anne Nicolaysen for preparing the histological cross-sections and to Dr R.Ding for useful comments on the manuscript. Supported by a grant from The Nordic Insulin Fund.

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