Acta physiol. scand. 1971. 81. 557-564 From the Department of Pharmacology, University of Lund, Sweden

Action Potential Generation in Denervated Rat Skeletal Muscle

I. Quantitative Aspects

BY

PAUL REDFERN] and STEPHEN THESLEFF

Received 14 Oktober 1970

Abstract

REDFERN, P. and S. THESLEFF. Action potential generation in denarvated rat skeletal muscle. I . Quantitative aspects. Acta physiol. scand. 1971. 81. 557-564.

Action potential generation was studied at various periods up to one week after denervation i n individual muscle fibres of the extensor digitorum longus muscles of the adult rat. To allow a comparison of action potential generation at various stages of denervation, it was necessary to establish adequate conditions for spike generation. I t was found that when fibres were locally polarized to a level of - 90 to - 100 mV, and the external calcium concentrations was increased to 4 mM, the peak rate of rise and the overshoot of the action potential were maximal. Between 30 and 40 hrs following section of the motor nerve, the mean maximal rate of rise of action potentials, recorded under the aforementioned conditions, was reduced by about one third, and remained a t about this reduced level during the subsequent days. Two days after denervation the resting membrane potential was reduced from a mean of 82 mV in innervated muscle to a mean of 68 mV, and remained at about this level for the remaining 5 days studied. The electrical time constant and the input resistance of the muscle fibres gradually increased during the 7 days following denervation, the time constant by about 70 $6 and the input resistance by about 50 %. With anodal polarization in denervated muscle no significant correlation was found between the resting membrane potential and the maximal peak rate of rise of the spike. I t was concluded that denervation produces a genuine reduction in the rate of rise of the action potential in muscle fibre.

Following denervation marked alterations occur in the electrophysiological proper- ties of mammalian skeletal muscle. The main changes in the extensor digitorum longus (E.D.L.) muscle of the rat include a fall in the resting membrane potential and a gradual increase in membrane resistance and total capacitance. (Albuquerque and Thesleff 1969, Albuquerque and McIsaac 1970). The electrical and chemical excitability of the muscle membrane is also changed, spontaneous action potentials appear and a gradual increase in the area of the membrane sensitive to acetylcholine is observed (Ginetsinsky and Shamarina 1942, Axelsson and Thesleff 1959, Miledi 1960). 1 Present address: Department of Anaesthesia, P. 0. Box 147, University of Liverpool, Liver- pool, England.

557

558 PAUL REDFERN AND STEPHEN THESLEFF

I t has been shown (Albuquerque and Thesleff 1968) that action potential genera- tion is impaired in muscles denervated 7-10 days, a t which time the rate of rise of the action potential is reduced. I t was not clear, however, whether the observed decrease in excitability was secondary to the reduction in the resting membrane potential, or attributable to other factors. The time course of the onset of the reduc- tion in electrical excitability was also unknown, and it was of interest to compare it with the appearance of the other changes in the membrane following denervation.

Experiments were therefore performed in which the resting membrane potential, the input resistance, the electrical time constant and action potential generation were measured in the E.D.L. muscle of the rat at various periods following dener- vation. To reduce the complication of muscle fibre atrophy, the investigation was limited to the first seven days following denervation.

Methods The E. D. L. muscle of male Wistar rats with a body weight of 18&220 g was denervated unilaterally, under ether anesthesia, close to the knee joint, about 5 mm from the entry of the nerve into the muscle. At various intervals following nerve section, the denervated muscle and its contralateral innervated control were removed and mounted together in an organ bath. The bath fluid had the composition described by Liley (1956) except that the con- centration of calcium was increased to 4 mM by the addition of calcium chloride, (see results), and was bubbled with 5 % COs in oxygen, giving the fluid a p H between 7.0 and 7.3. The temperature of the bath was maintained at 29" C f 0 . 5 " C. The membrane potential during rest and impulse activity was recorded from the surface fibres of the muscle using conventional glass capillary microelectrodes with a resistance of 4 to 10 Mohms, the input capacitance of the recording circuit with the microelectrodes was 5-10 pF.

T o generate and record the action potential, two microelectrodes were inserted into the fibre about 50 ,um apart, one electrode, connected through a 100 Mohm resistance, was used to pass current, and the other to record the potential change. Constant anodal current in steps of varying magnitude was passed through the membrane for 30 to 60 sec, a time found to give steady-state conditions. This current terminated in a 5 msec cathodal shock, adjusted in each case to produce an action potential with 1-3 msec latency. The technique of stimula- tion and recording, generally produced little fall in resting potential, and several spikes could be recorded from the same fibre. The rate of rise of the action potential was obtained by the use of a R C derivating circuit (100 pF; 100 Kohms).

The input resistance of single fibres was measured using pulses of varying intensity and 200 msec duration, covering a range of anelectrotonic currents causing a potential change between 2 and 10 mV. The average result of such a series was used. The electrical time constant of the membrane was obtained by measuring the time required for the membrane potential change to reach 83 % of its plateau value (Boyd and Martin 1959).

A series of muscles were prepared by soaking the pair for 1 hour in a bath solution made hypertonic by the addition of 400 mM glycerol, and then returning the muscles to isotonic solution. This procedure, described by Howell and Jenden (1967) is known to disrupt the sarcotubular system in frog muscle (Eisenberg and Eisenberg 1968), and thereby to abolish excitation-contraction coupling (Gage and Eisenberg 1969).

Results Resting membrane potential. On the second day following denervation the resting membrane potential was reduced from a mean of 82 mV to a mean of 68 mV and this value was little changed for the remaining 5 days of the period examined, as shown in Fig. 1. The resting membrane potential appeared to fall rather abruptly, at about 30 hrs after denervation.

ACTION POTENTIALS IN DENERVATED MUSCLE 559

Fig. 1 . The effects of denervation on the resting membrane poten- tial of individual fibres of the E.D.L. muscle of the rat. The open circles indicate the mean 2 S.D. of values in denervated muscles, and the closed circles indicate the mean k S.D. of values from the contralateral innervated muscles. The abscissa shows the period of denervation on two dif- ferent time scales. The mean? S.D. of the resting membrane po- tential of all innervated muscie fibres is indicated by the dotted line and values at zero time. Each mean was obtained from at least two muscles, and the value next to. each point is the number of fibres examined, day5 hours dOy5

Effects of anodal polarization on action potential. The marked difference in resting membrane potential between innervated muscle and muscle denervated for 2 or more days, made direct comparison of their action potentials meaningless, since spike generation is dependent on the level of polarization of the cell (Hodgkin and Huxley 1952). Anodal polarization restores the excitability of nerve and muscle depolarized by a variety of means (Frankenhaeuser and Hodgkin 1957). This technique was therefore adapted to the present study of action potential generation.

Anodal currents allowed the membrane potential of the muscle cell to be locally set to values up to -120 mV, and the relationship between this potential, and the local generation of action current, to be determined. Responses were measured and expressed as the maximal rate of rise. The maximum rate of rise reflects inward current at that moment, and in the absence of voltage clamp conditions, this is probably the most meaningful index of spike generation.

Representative action potentials in an innervated and a 4 day denervated muscle at various levels of membrane polarization are shown in Fig. 2. In both innervated and denervated muscle, maximal peak rates of rise were observed when the mem- brane was polarized to -80 to -100 mV. Further polarization failed to increase the rate of rise of the action potential, and often reduced it. At potential levels below -75 mV, action current was greatly reduced (Fig. 3 ) . Glycerol treatment of muscle, which abolished excitation-contraction coupling, and allowed the re- cording of repeated spikes uncomplicated by mechanical response, revealed the same essential relationship between the level of polarization and the maximal rate of rise of the spike, showing that this relationship was not influenced by mechanical twitching (Fig. 4).

Since peak responses were obtained at polarization levels of -80 to - 100 mV all subsequent recordings of action potential in both control and denervated muscle were carried out at local potential between -90 and -100 mV.

560 PAUL REDFERN AND STEPHEN THESLEFF

8 p

7 -

6 -

5 -

4 -

3 -

2 -

I '

50 mV

500 '4,

Fig.

1 b I

V/SeCXlO'

t t 1 8 I '

I . i I " I I 0

I

0' Qo Q5 ;o ;5 e'o e'5 ;o 9'5 ti0 1;)s mV

Fig. 3 Fig. 2. Cmsecutive action potentials (upper traces) and their first derivatives (lower traces) from an individual fibre of an innervated E.D.L. muscle (left hand record) and from a fibre of a 4 day denervated E.D.L. muscle (right hand record). The fibres were locally polarized to the potential levels indicated by the numbers, and the gap in the recordings of the action potentials shows the zero Fotential of the cell. The duration of the stimulating current pulse was 5 msec. Fig. 3. The maximal rates of rise (ordinate) of action potentials at various levels of local membrane polarization (abscissa) in 4 day denervated muscles (open circles) and in the contralateral innervated muscles (closed circles). Each value is the meankS3.D. of 5 to 16 measurements in two E.R.L. muscles.

External calcium is known to play an important part in membrane excitation. The sodium activation mechanism is in addition to membrane polarization determined by the calcium ion concentration, among other things (Frankenhaeuser and Hodgkin 1957). To establish the adequate concentration of calcium for action potential generation in both innervated and denervated muscle, experiments were made in solutions containing 2,4, 6 and 8 mM calcium. No significant differences in maximal rate of rise of the action potential was observed at these calcium concentrations, but the mean values indicated that maximal rates of rise were obtained in the presence of 4 mM calcium. This concentration was, therefore used in all experiments.

ACTION POTENTIALS IN DENERVATED MUSCLE 56 1

Fig. 4. Consecutive and superimposed action poten- tials and their first derivatives from a single fibre of an innervated, glycerol treated E.D.L. muscle. As shown by the record, the level of membrane anodal polarization was changed between each action po- tential. The broken line indicates the zero potential level of the fibre.

- 2 msec

Effects of denervation on action potential. Having established the aforementioned conditions for spike generation in innervated muscle, it became possible to make a quantitative study of the effects of denervation on the action potential. As shown in Fig. 5, the average maximal rate of rise of the action potential was reduced on the second day following denervation from 630 V/sec to 370 Vlsec. During the subsequent days, the rate of rise of the spike remained at about this reduced level. The amount by which the spike exceeded zero membrane potential, i.e. overshoot, was not as markedly changed by denervation. A closer examination of the time course of the fall in the rate of rise of the action potential showed that it occurred quite rapidly between 30 and 40 hrs of sectioning the nerve.

Denervation not only reduced the rate of rise of the action potential, but also reduced its rate of repolarization and prolonged its duration (Fig. 2 ) . These changes were already apparent two days after denervation, and were not markedly altered during the subsequent 5 days.

The possibility that the fall in resting membrane potential was the direct cause of the observed reduction in action current was examined by analysing the data for muscles denervated for 2 or more days for a possible correlation between the resting membrane potential, and the maximal rate of rise of the spike. There was no significant relationship (correlation coefficient 0.056). Glycerol treatment of muscles is known to produce a variable fall in the resting membrane potential (Gage and Eisenberg 1969) and in our study the mean resting membrane potential of 136 in- nervated skeletal fibres following glycerol treatment was reduced to 63 f 1.1 mV. Among these glycerol treated muscle fibres, there was also no significant correlation between the resting membrane potential and the maximal rate of rise of the action potential (correlation coefficient 0.008).

9-713003. Ada phyriol. rcand. Vol. 81: 4 .

562 PAUL REDFERN AND STEPHEN THESLEFF

4-- 5 6 1 days hours do ys

Fig. 5. The maximal rate of rise of action potential (ordinate) in muscles denervated (open circles) for the time shown by the ab- scissa, and in the contralateral in- nervated muscles (closed circles). Each value is the meanfS.D. of measurements from a t least 2 muscles, and the adjacent figure indicates the number of fibres ex- amined. The meanfS.D. for all the innervated muscles is indica- ted by the values at zero time, and by the dotted line. All re- cordings were made in fibres po- larized to - 90 to - 100 mV.

I n the present recordings, no effort was made to distinguish between end plate region and the rest of the fibre and, therefore it is not possible to state whether the observed change in action potential generation developed initially in relation to the end plate region, or simultaneously in the whole fibre.

Effects of denervation on input resistance and electrical time constant. The ob- served decrease in electrical excitability following denervation, could reflect the lengthening of the electrical time constant, observed in muscles following denervation (Albuquerque and Thesleff 1968). Table I shows the mean values of input resistance and electrical time constant of the fibres of innervated and denervated muscles. Both properties increase gradually during the 7 days following denervation, the time constant by about 70 % and the input resistance by about 50 %. A similar gradual increase in these two parameters has been reported by Albuquerque and McIsaac ( 1970).

TABLE I. Influence of denervation on mean f S.D. membrane time constant and meanfS.D. in- put resistance of single fibres of E.D.L. muscle of the rat. Figures within parenthesis are the number of fibres examined.

Days of denervation Membrane time constant Input resistance m e c Mohm

0 3.0f0.52 (125) 0.49&0.127 (55)

3~7h0.37 ( 27) 0.50f0.034 (10) 3 4 4.550.55 ( 23) 5 0.66&0,142 (10) 6 5 . 2 0 . 8 7 ( 20) 0.68&0.115 (19) 7 5.2&0.46 ( 19) 0.72k0.144 (22)

2 2.750.73 ( 19) - -

563 ACTION POTENTIALS IN DENERVATED MUSCLE

Discussion In order to make a quantitative comparison of action potentials in different muscles, it is necessary to establish either identical or optimal conditions for spike generation. The observed fall in resting membrane potential following denervation precluded a direct comparison between the action potential of innervated and denervated muscle. In the absence of adequate techniques for voltage clamping of muscle fibres, it was decided to use anodal polarization. With this procedure, the membrane potential of the cell is locally changed at the site of the current electrode and from that point exponentially decaying over an area determined by the electrical length constant of the fibre, which in both innervated and denervated E.D.L. muscles is about 0.5 mm (Albuquerque and Thesleff 1968). Despite this limitation to the technique, the fact that the action current, as evidenced by the maximal rate of rise of the spike, reached a plateau in both innervated and denervated muscles, indicates that suffi- cient polarization levels had been reached. A calcium concentration of 4 mM was found adequate for spike generation in innervated and denervated muscle.

A comparison of the action potential under the aforementioned conditions, showed that the rate of rise and the amount exceeding zero membrane potential were always reduced in muscle denervated for 2 or more days. Since with the technique of anodal polarization, no correlation was found between resting membrane potential and maximal rate of rise of the action potential in depolarized fibres, it is unlikely that the fall in rate of rise following denervation was secondary to the reduction in rest- ing membrane potential. I t can be concluded, therefore, that denervation produces a genuine reduction in the rate of rise of the action potential in the muscle fibre. It is of interest, that this reduction in spike generation, and also the fall in resting membrane potential, occurs abruptly at 30 or 40 hrs after denervation, shortly after the time at which it is known that transmitter release from the degenerating nerve terminals stops (Miledi and Slater 1968, Albuquerque and McIsaac 1970). I t should also be mentioned that at this time the acetylcholine sensitive area of the muscle fibre membrane starts to spread.

The fall in resting membrane potential and in the rate of rise of the action poten- tial are the earliest post-denervation changes in muscle. Since these changes affect the entire muscle cell, it seems reasonable to assume that they are the result of rapid- ly developing structural changes in the cell membrane.

The reduction in the maximum rate of rise of the spike following denervation could be due to the observed lengthening of the electrical time constant. However, the change in the time constant following denervation was not coincident with the fall in the rise rate of the spike. Another explanation would be that denervation reduced the number or the efficiency of the membrane sites for action potential generation. The finding that the action potential following denervation, becomes partly resistant to the blocking action of tetrodotoxin (Redfern, Lundh and Thesleff 1970) , indicates that qualitative changes occur in the membrane sites responsible for spike generation.

564 PAUL REDFERN AND STEPHEN THESLEFF

This study was supported by a research grant from the Swedish Medical Research Council (B70-14X-738-05B), Stockholm, Sweden. P. A. Redfern was in receipt of a Clinical Research Fellowship from the Wellcome Trust during the course of this study.

References ALBUQUERQUE, E. X. and R. I. MCISAAC, Fast and slow mammalian muscles after denervation.

Exp. Neurol. 1970. 26. 183-202. ALBUQUERQUE, E. X. and S. THESLEPF, A comparative study of membrane properties of in-

nervated and chronically denervated fast and slow skeletal muscles of the rat. Acta physiol. scand. 1968. 73. 471480.

AXELSSON, J. and S. THESLEFF, A study of supersensitivity in denervated mammalian skeletal muscle. I. Physiol. (Lond.) 1959. 147. 178-193.

BOYD, I. A. and A. R: MARTIN, Membrane constants of mammalian muscle fibres. J. Physiol. (Lond.) 1959. 147. 450-457.

EISENBERO, R. and R. S. EISENBERG, Selective disruption of the sarcotubular system in frog sartorius muscle. J. cell. Biol. 1968. 39. 451-467.

FRANICENHAEUSER, B. and A. L. HOWKIN, The action of calcium on the electrical properties of squid axons. J. Physiol. (Lond.) 1957. 137. 217-244.

GAOE, P. W. and R. S. EXSENBERG, Action potentials, afterpotentials, and excitation-contraction coupling in frog sartorius fibres without transverse tubules. J . gen. Physiol. 1969. 53. 298- 310.

GINETSINSKY, A. G. and N. M. SHAMARINA, Tonomotornyi fenomen v denervirovannoi myshtse. Us$. Sovrem. Biol. 1942. 15. 283-294.

HODGKIN, A. L. and A. F. HUXLEY, The dual effect of membrane potential on sodium con- ductane in the giant axon of Loligo. ]. Physiol. (Lond.) 1952. 116. 497-506.

HOWELL, I. N. and D. I. JENDEN, T-tubules of skeletal muscle : morphological alterations which interrupt excitation-contraction coupling. Fed. Proc. 1967. 26. 553.

LILEY, A. W., An investigation of spontaneous activity of the neuromuscular junction of the rat. 1. Physiol. (Lond.) 1956. 132. 650-666.

MILEDI, R., The acetylcholine sensitivity of frog muscles after complete or partial denervation. J. Physiol. (Lond.) 1960. 151. 1-23.

MILEDI, R. and C. R. SLATER, Electrophysiology and electron microscopy of rat neuromuscular junctions after nerve degeneration. Proc. roy. SOC. B. 1968. 196. 289-306.

REDFERN, P., H. LUNDH and S. THESLEFF, Tetrodotoxin resistant action potentials in dener- vated rat skeletal muscle. Europ. 1. Pharmacol. 1970. 11. 263-265.