Repetitive action potentials induced in chloride-free solution: Effect of denervation

EXPERIMENTAL

NEUROLOGY

Repetitive

91,409-422

Action Potentials Induced in Chloride-Free Solution: Effect of Denervation BASILIO

Centro

de Investigaciones Donato Received

(1986)

ARISTIDIS

KOTSIAS’

Medicas A. Einstein and Institute de Investigaciones Alvarez 3150, 1427 Buenos Aires, Argentina

December

12, 1984; revision

received

September

Mdicas,

27, 1985

Isometric mechanical activity and action potentials registered with intracellular microelectrodes were studied in innervated and denervated fibers of the soleus muscle of the rat in normal and chloride-free solutions. The chloride-free solution promoted in both innervated and denervated fibers an increment in the resting membrane potential. The innervated muscles showed long mechanical relaxation and repetitive action potentials after a single depolarizing pulse. On the contrary, denervated muscles were resistant to show mechanical and electrical changes in the chloride-free medium. Spontaneous and evoked action potentials from innervated muscle fibers were abolished by tetrodotoxin. The evoked action potentials generated in denervated fibers had a slower time course and were resistant to tetrodotoxin. After 7 to 10 days of denervation the input resistance was increased by about 30%. Substitution of chloride with sulfate resulted in a 150% increase in input resistance of innervated muscle fibers and 80% in denervated preparations. Alterations in the ionic conductances, a decrease in the maximum rate of rise of the action potentials, and changes in the sodium current kinetics could be the main factors for the absence of repetitive action potentials in denervated fibers exposed to the chloride-free medium. 0 1986 Academic pms, hc. INTRODUCTION

In the last years several methods that induced muscular signs resembling myotonia have been described: (i) administration of (a) hypocholesterolemic drugs, i.e., 20,2Sdiazacholesterol (20,25DC) (18), (b) aromatic carboxylic acids such as 2,4-dicholorophenoxy acid (2,4-D) (33), and (c) diuretics (3); (ii) application of emetine in the motor nerve of the muscle (10); and (iii) Abbreviations: AP-action potential, TTX-tetrodotoxin. ’ I thank Dr. S. Muchnik and Dr. R. A. Venosa for helpful discussions. This work was supported by grants from the CONICET of Argentina and Muscular Dystrophy Association, U.S.A. 409 0014-4886/86

$3.00

Copyright 0 1986 by Academic Ress, Inc. All rights of reproduction in any form reserved.

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replacement of the chloride ions in the normal solution by an impermeant anion such as sulfate or methylsulfate (1, 16) [for reviews see (32, 36)]. Those procedures provide useful tools for studying and testing the different hypotheses that have been proposed to explain the mechanisms of myotonic signs. Myotonia has been considered to be the result of a muscular defect independent of the nervous system (24). However, there is controversy regarding the involvement of the nervous system in the initiation of experimental myotonia. Thus some authors (5, 11, 33) have shown that denervation of the muscle produced a resistance to the development of electrical or mechanical signs of myotonia in response to administration of 20,25-DC or 2,4-D. On the basis of those studies it was proposed that innervation is necessary in maintaining the membrane capable of generating repetitive electrical activity. In contrast, other investigators (14) found that rat muscles denervated for more than 10 days and treated with 2,4-D showed a prolonged relaxation which is one of the characteristics of the myotonic syndrome. This paper was concerned with the role of innervation on the repetitive activity induced in chloride-free solution: the relationship between electrical and mechanical activity was studied. Evidence is presented which indicates that denervation of muscle fibers results in a resistance to generate action potentials (APs) when chloride ions are replaced by sulfate ions. METHODS Experiments were conducted in rat (Wistar) soleus muscles in vitro at 32°C. Denervation was carried out by removing 5 mm of the right sciatic nerve under ether anesthesia: after 7 to 11 days the muscles were dissected and studied. Mechanical Measurements.The muscle was placed in an insulated chamber and connected to a Grass force transducer. Massive stimulation through platinum electrodes was used. The duration of the pulse was 0.3 ms. The isometric responses elicited at different frequencies of stimulation were registered using a Gould recorder. In these experiments, d-tubocurarine ( 10e5 g/ml) was added to the solutions. Action Potential Measurements.The muscle was mounted into an insulated chamber and superfused at a rate of 1 to 2 ml/min with saline. The fiber was impaled with two microelectrodes (6 to 15 MS2,3 M KCl), a recording and a stimulating electrode, inserted at about 100 pm from one another. In some cases the fiber was hyperpolarized to -90 mV with anodal current through the stimulating electrode for 5 to 10 s before a depolarizing pulse of 2- to 3- ms duration via a WPI electrometer (2, 11, 34, 38). The output of the

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electrometer was connected to a Tektronix oscilloscope. The AP and its first derivative (F. Haer & Co.) were displayed on the oscilloscope. Input Resistance.We measured the input resistance of the fiber with a single microelectrode technique. The electrode filled with 2 M K-citrate was connected to a high-input impedance electrometer (WPI & Co.) which allowed us to record the changes in membrane potential and inject current simultaneously via a subtracting circuit. Prior to penetration the voltage subtracting circuit was balanced delivering a current step (50 nA). Occasional slight adjustments were necessary when the fiber was impaled. The null point was easily determined because of the long time constant of the recording circuit. The null point was rechecked again after withdrawal of the microelectrode. The input resistance of the fiber was taken as the ratio between the change in membrane potential and the current applied. In the absence of chloride ions the excitability of the fiber was increased (see Results). To avoid the generation of repetitive APs, tetrodotoxin (TTX, 10e6 M) was added to the solutions. Pulses of 100 to 200 ms were used. The normal Ringer’s solution contained (a): NaCl 135, KC1 5, CaQ 2, MgC& 1, NaHC03 20, Na2HP04 1, glucose 11. The pH was 7.2 to 7.4 when bubbled with 95% OZ-5% COZ. Isotonic sulfate solution: chloride-free solution was prepared with sulfate replacing chloride ions and following the procedures of Hodgkin et al. (22). RESULTS Mechanical Activity. Figure 1 shows the mechanical response in normal (A-C) and chloride-free solution (E-H) elicited at different frequencies of stimulation. The twitch of soleus muscle in normal solution did not present the potentiation at low frequencies of stimulation characteristic of fast muscles (staircase phenomenon). It is seen that at these frequencies (3 to 10 Hz) the muscle responded with individual twitches. When normal saline was replaced by a solution without chloride ions there was a gradual increment in the twitch tension (Fig. 1D). In about 5 to 15 min the mechanical responses were characterized by a long relaxation accompanied by fusion of individual twitches. Sometimes the tension developed in response to a single stimulus approached tetanic tension and was maintained for several seconds. After 70 to 90 min the muscle tension began to decrease with a slow time course presumably due to deterioration of the preparation. Figure 2 depicts response of a ‘I-day denervated muscle in normal and chloride-free solutions. When chloride ions were replaced with sulfate and in contrast to innervated muscles, the twitches did not present the long relaxation time and remained unfused (six experiments). We tested muscles to 11 days of denervation. Longer periods of denervation resulted in severe atrophy of

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FIG. 1. Twitches of an innervated soleus muscle in normal Ringer’s (A-C) and in a chloridefree solution (D-H) elicited at 3 (A, F), 5 (B, G), and 10 Hz (C, H). The arrow in D indicates the moment when normal solution was replaced by a chloride-free solutin. E shows a typical twitch with a long relaxation time. Horizontal calibration: 2 s (C, H), 4 s (B, G), 10 s (A, E, F), 400 s (D); vertical calibration: 12 g.

the muscle fibers with proliferation of connective tissue which made difficult the impalement of the fiber with microelectrodes. The effects of chloride-free solution could be reversed by washing with normal Ringer’s solution (three experiments).

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FIG. 2. Twitches in a soleus muscle denervated for 7 days in normal Ringer’s (A-C) and chloride-free solution (D-G) elicited at 3 (A, E), 5 (B, F), and IO Hz (C, G). The arrow in D indicates the changes of the external solution. Vertical calibration: 12 g; horizontal calibration: 2 s (C, G), 4 s (B. F), 10 s (A, E), 400 s(D).

Resting Membrane Potentials. Figure 3 presents the resting membrane potential values (Vm) obtained from innervated and denervated muscle fibers (7 to 10 days) in normal and chloride-free solution. In the normal solution the Vm of denervated fibers was 10 mV lower than the values from control preparations [see (2, 19, 35)]. In the chloride-free solution the innervated fibers (Fig. 3, filled symbols) showed a transient depolarization of 10 mV followed by hyperpolarization that attained a steady-state value. The Vm of denervated fibers increased 6 to 9 mV (Fig. 3, open symbols) when chloride ions were replaced by sulfate and lacked the transient depolarization observed in the innervated fibers.

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FIG. 3. Resting membrane potential (mV) in normal Ringer’s (NR) and chloride-free solution (5 K-S) in innervated (filled symbols) and denervated muscle fibers (7 to 10 days, open symbols). Each point represents the average of 10 to 15 impalements.

SpontaneousElectrical Activity. Fibrillation activity could be recorded in vivo in all muscle fibers denervated for more than 4 days. When the muscle was removed and placed in a bath with normal solution the fibrillation activity disappeared in a few minutes (38). Spontaneous APs were recorded in vitro in both innervated and denervated muscles in chloride-free solution. The surface cells were randomly impaled and the presence of spontaneous activity noted during 30 to 60 s. The percentage of innervated fibers that exhibited electrical activity was higher than in the denervated fibers; 40 of 50 innervated fibers showed spontaneous APs. These APs were preceded by a prepotential with a slow rate of rise and most of them had overshoots. The amplitude of these responses decreased during the first 2 min of recording. In 63 fibers denervated for 7 to 10 days, 38 of them presented spontaneous APs. The APs were present in denervated fibers with different Vm values although the amplitude of the spikes decreased in fibers with low Vm values. The impalement of the fiber with a second microelectrode did not increase the frequency of the spontaneous APs; that was the rule with the innervated fibers. Evoked Action Potentials. To minimize the effects of variations in the Vm upon the APs recorded in innervated and denervated fibers, the Vm of the fibers was set to a local value of -90 mV by passing a constant current through the membrane (see Methods). Figure 4A shows an AP and its time derivative in normal Ringer%. The record from a denervated fiber is shown in Fig. 5A. It is clear that the AP generated in the denervated fiber had a slower time

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FIG. 4. Evoked APs in innervated fibers in normal Ringer’s (A) and chloride-free solution (BD). C depicts the typical behavior of an innervated fiber in the absence of chloride ions. D: response of a fiber in chloride-free solution when tetrodotoxin (TTX) was added to the bath. In A, B, and D the fibers were hyperpolarized to -90 mV (see Methods). Action potential spikes retouched. Vertical calibration: 40 mV and 400 V/s (A), horizontal calibration: 2 ms (A, B, D), 100 ms (C).

course and was smaller than the AP registered in the innervated fiber. This is in agreement with previous results (2, 11). Anode break APs were present in denervated preparations. Table 1 summarizes the AP characteristics from innervated and denervated fibers exposed to different solutions. Figure 4C presents a record from an innervated fiber immersed in chloridefree solution. These records were obtained in fibers not hyperpolarized and at a slow time base. The cessation of the single stimulus gave rise to afterdis-

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FIG. 5. Evoked APs in denervated fibers (10 days) in normal Ringer’s (A) and chloride-free solution (B-E). Records in A, B, D, and E were taken from fibers hypexpolarized to -90 mV. C: typical response of the denervated fiber (not hyperpolarixd) in chloride-free solutions. E shows a TTX-resistant AP. Action potential spikes were retouched. Vertical calibration: 40 mV and 400 V/s (A); horizontal calibration: 2 ms (A, B, E), 100 ms (C, D).

Ringer’s

Normal

days)

Potential

and Action

-57.3 -64.2

in Normal

13.6 f 5.2 8.7 z!z 6.0 18.5 + 7.5

k 3.5

f 4.5

14.3 + 4.6 24.5 + 6.5

18.5 + 5.0

Overshoot WV)

Potentials

f 2.6

+ 4.5 k 6.5

-63.1 -74.0

-58.2

+ 3.5

-65.2

Vm (mV)

(Vm)

1.0 + 0.1 1.1 + 0.2

1.0 f 0.2 245 zk 75 291 + 41

241 t 34

rate of rise, -dV/dt:

114 f 21

115 k 28

121 f 22

147 f 21 176 f 44

280 + 51 379 + 65

0.9 2 0. I 0.9 + 0.2

-dV/dt W/s)

163 f 28

+dV/dt (V/s)

Ca), and Chloride-Free

potentials

(0.5 mM

Action

Calcium

326 f 43

Low

0.9 f 0.2

Duration (ms)

Ringer’s,

I

0 Values are X f SD. Duration of the action potential was measured at -40 mV. +dV/df: maximum The fibers were hyperpolarized to -90 mV before applying the depolarizing pulse (see Methods).

O-Cl

0.5 Ca

(7-9

Ringer’s

Membrane

Denervated

0.5 Ca O-Cl

Normal

Innervated

Resting

TABLE (O-Cl)’

maximum

rate of fall.

2131

2125

O/26

O/23 23127

O/28

No. of fibers with rep. activity

Solutions

3 5

R

:

?

2

2

3 =i

E

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charge which consisted of a train of APs of gradually decreasing amplitude and rates of rise. Fibers displaying repetitive activity occurred in about 85% of the preparations studied (see Table 1). Similar results were obtained using fibers hyperpolarized to -90 mV. In chloride-free solution the most significative difference observed between the innervated and denervated fibers was the lack of repetitive generation of APs after a single stimulus (Fig. SC): a single depolarizing pulse generated only one AP which was followed by a long-lasting after potential characteristic of denervated muscles (37). We considered the possibility that the lower Vm of denervated fibers might hinder the generation of repetitive APs. To study this possibility we hyperpolarized the fibers to -90 mV prior to application of a single depolarizing pulse. Figure 5D shows an experiment of this type. It is evident that the hyperpolarized fiber failed to generate repetitive APs in the absence of chloride ions. The concentration of ionized calcium in the chloride-free solution used in our experiments was about 1 mM (22). A possibility to be considered is that the repetitive firing exhibited by innervated muscles in the absence of chloride ions was facilitated by a shifting of the inward current toward more negative potentials (17, 2 1) or as it was ascribed in Aplysia neurons to a lowering of potassium conductance secondary to a decrease in internal calcium concentration (15). In this regard, a Ca-dependent K conductance was reported in rat myotubes (30) and rat soleus muscle (8). To investigate this point, experiments were done in innervated and denervated muscles bathed in a chloride solution with 0.5 rnMCaC1z (30 min of equilibration in this solution). In this lowered calcium concentration a single stimulus applied to an innervated or denervated fiber usually gave rise to a single AP. No evidence of repetitive APs were noted. The experiments are summarized in Table 1. In addition, control experiments with muscles exposed to low calcium concentration failed to demonstrate changes in the time course of the twitches. In innervated fibers, 10T6 MTTX blocked the spontaneous and the evoked APs (Fig. 4D, 19 fibers); in denervated fibers, TTX blocked the spontaneous APs (45 fibers), however many fibers continued to generate evoked APs but with a slower time course than in the absence of the drug (Fig. 5E). The blocking effect of TTX on the electrical activity is in agreement with results by D’Alonzo et al. (11) and Gruener et al. (20) who demonstrated that TTX blocked 20,25-DC myotonia and the electrical activity in myotonia congenita. Input Resistance. To obtain information about the chloride conductance in denervated fibers we measured the input resistance of the fibers in normal and chloride-free solutions. In chloride-containing solution the input resistance in innervated fibers was 650 + 280 KQ (X + 1 SD, four muscles, 45 fibers). After 7 to 10 days of

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denervation the input resistance was increased by about 30%: 850 f 340 KQ (five muscles, 59 fibers). In an innervated fiber substitution of chloride with sulfate resulted in a 150% increase in input resistance: 1500 + 500 KQ. In 7- to 1O-days denervated fibers the increment of resistance in chloride-free medium was 80%: 1520 + 490 KQ. Thus, the ratio between the input resistance values in denervated to innervated fibers was close to one. These results are in agreement with other reports (27, 37). The increment in input resistance in chloride-free solution could be reversed by washing with normal solution (six experiments). DISCUSSION The following electrophysiologic changes characterize an innervated fiber exposed to a chloride-free medium: (a) The Vm is increased approximately 10 mV. Dulhunty (12) studied the effects of chloride concentration on Vm and suggested that a reduction in the internal concentration accounts for the hyperpolarization of the fibers and the time taken for Vm to reach a steadystate value after reduction of chloride ions is a consequence of the time taken for the internal concentration of chloride to change. (b) The electrical input resistance is increased by about 150% as a consequence of the dramatic reduction in chloride conductance. (c) When the fiber is stimulated with a single stimulus it gives rise to a train of APs. In a normal medium the high chloride conductance stabilizes the response (29). The repetitive discharge and its related long mechanical relaxation elicited by a single pulse in innervated fibers exposed to a chloride free solution could be considered as a simple model of myotonia. Several factors have been proposed to contribute to myotonic signs: low chloride conductance (I, 26), alterations in the kinetics of sodium conductance (4, 25) or K conductance (28), or changes in the physical properties of the membrane (7). The main result of the present work in the absence of repetitive activity after a single pulse in denervated fibers in chloride-free solution. This result is somewhat puzzling in light of the observation that denervated rat muscle in vivo is characterized by spontaneous electrical activity indicating an enhanced excitability. Several possibilities may be suggested as explanations for the absence of repetitive APs in denervated fibers exposed to a chloride-free solution: (i) We found that in denervated fibers substitution of chloride by sulfate resulted in a smaller increment in input resistance in comparison with the changes that occurred in innervated fibers. This is in agreement with previous authors (27,37) who found that chloride conductance of mammalian skeletal muscle decreased significantly after denervation as a consequence of the disruption

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of some trophic material transported through the axon (6). The reports concerning the effects of denervation on potassium permeability are not clear, depending on the methodologies employed and the time of denervation (6, 23, 27). Nevertheless, the &i:gx ratio is different in denervated muscle compared with innervated muscle. Thus, this ratio obtained in soleus muscle is reduced from 6.4 to 1.6 after 7 days of denervation (27). That denervated muscle is resistant to changes in external chloride concentration could be explained on this basis. (ii) In agreement with other workers we found that the time course of the AP in innervated fibers is faster than in the denervated preparations (2, 11, 38). D’Alonzo et al. (11) suggested that the reduction in the maximum rate of rise of the AP which reflects a reduction in the responsiveness of the membrane is the reason for the resistance of denervated muscle to 20,25-DC myotonia. Thus, the nervous system would influence the repetitive generation of APs of a muscle by controlling the characteristics of the responses. However, it should be mentioned that maximum sodium conductance in denervated fibers is similar to control values ( 13, 3 1). Pappone (3 1) observed in denervated fibers a shifting of the sodium inactivation curve in the negative direction. An interesting possibility is that this change in sodium current could be an important factor in the phenomenon described in this work: fewer sodium channels, at the end of the first response, would be available to generate a train of APs. An alternative explanation for the absence of repetitive activity in denervated muscle fibers could be that the long-lasting afterpotential presented in the fibers activates a particular type of potassium channels similar to those described in other tissues (9). The ensuing outward current through the K channels may balance the depolarization of the fiber and stabilize the Vm. Because we do not have data about the K channels, this is merely a speculation. Our results are an approximation to the problem. Further studies and voltage clamp experiments in isolated fibers will be necessary to clarify this matter. REFERENCES 1. ADRIAN, R. H., AND S. H. BRYANT. 1974. On the repetitive discharge in myotonic muscle fibres. J. Physiol. (London) 240: 505-S 15. 2. ALBUQUERQUE,E. X., AND S. THESLEFF. 1968. A comparative study of membrane properties of innervated and chronically denervated fast and slow skeletal muscles of the rat. Acta Physiol. &and. 73: 471-480. 3. BRETAG, A. H., S. R. DAWE, D. I. B. KERR, AND A. G. MASKWA. 1980. Myotonia as a side effect of diuretic action. Br. J. Pharmacol. 71: 467-47 1. 4. BRYANT, S. H., AND T. E. DE COURSEY.1980. Sodium currents in cut skeletal muscle fibres from normal and myotonic goats. J. Physiol. (London) 307: 3 l-32P. 5. CACCIA, M. R., A. BOIARDI, L. ANDREUSSI, AND F. CORNELIO. 1974. Nerve supply and experimental myotonia in rats. J. Neural. Sci. 24: 145-150. 6. CAMERINO, D., AND S. R. BRYANT. 1976. Effects of denervation and colchicine treatment on the chloride conductance of rat skeletal muscle fibres. J. Neurobiol. 7: 22 l-228.

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15: 466-475.

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200: 437-439.

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30. PALLOTA, B. S., K. L. MAGLEBY, AND J. N. BARRETT. 1981. Single channel recordings of Ca2+-activated K+ currents in rat muscle cell culture. Nature 293: 47 l-474. 3 I. PAPPONE, P. A. 1980. Voltage-clamp experiments in normal and denervated mammalian skeletal muscle fibres. J. Physiol. (London) 306: 377-410. 32. PETER, J. B., AND D. S. CAMPION. 1977. Animal models of myotonia. Pages 739-746 in L. P. ROWLAND, Ed. Pathogenesis of Human Muscular Dystrophies. Excerpta Medica, Amsterdam. 33. RANISH, N. A., W. D. DETTBARN, AND V. IYER. 1977. The influence of nerve stump length on (2,4-dichlorophenoxy) acetic acid-induced myotonia. Exp. Neurol. 54: 393-396. 34. REDFERN,P., AND S. THESLEFF. 1977. Action potential generation in denervated rat skeletal muscle. II. The action of tetrodotoxin. Acta Physiol. &and. 82: 70-78. 35. ROBBINS, N. 1977. Cation movements in normal and short-term denervated rat fast twitch muscle. J. Physiol. (London) 271: 605-624. 36. RUDEL, R. AND F. LEHMANN-HORN. 1985. Membrane changes in cells from myotonia patients. Physiol. Rev. 65: 310-356. 37. THESLEFF, S. 1963. Spontaneous electrical activity in denervated rat skeletal muscle. Pages 4 l-5 I in E. GUTMANN AND P. HNIK, Eds., Neuromuscular Functions. Elsevier, Amsterdam. 38. THESLEFF, S., AND M. R. WARD. 1975. Studies on the mechanism of fibrillation potentials in denervated muscle. J. Physiol. (London) 244: 3 13-323.