336 J. Phy8iol. (1961), 156, pp. 336-343 With 6 text-ftgures Printed in Great Britain AN ELECTROPHYSIOLOGIC STUDY OF THE NEURO- MUSCULAR JUNCTION IN MYASTHENIA GRAVIS BY 0. DAHLBACK, D. ELMQVIST, T. R. JOHNS,* S. RADNER AND S. THESLEFF From the Departments of Pharmacology, of Thoracic Surgery, and of Medicine, University of Lund, Sweden (Received 5 December 1960) After a study of some of the electrophysiological properties of the neuromuscular junction in isolated intercostal muscles obtained from patients with no known neuromuscular disorder (Elmqvist, Johns & Thesleff, 1960), a similar study was undertaken in patients with myasthenia gravis. The intercostal muscles were obtained by biopsy under regional anaesthesia from six patients with chronic and non-localized myasthenia gravis. Immediately upon removal the muscles were studied by the use of intracellular electrodes. Evidence will be presented for a pre-junctional deficiency of transmitter formation or transmitter release. METHODS The group of six patients with chronic active non-localized myasthenia gravis included one male and five females, with ages from 18 to 57 years. No patient was selected in whom there was doubt regarding the diagnosis. The study was not done on patients with acute severe myasthenia gravis at the time of the biopsy. One patient had had a thymectomy followed by a marked remission. Intercostal muscles, usually the external, were obtained from the 5th to 7th intercostal spacesinthemid-axillaryline. The muscleswere carefully dissectedunderregionalanaesthesia, and then studied under conditions similar to those described by Elmqvist et al. (1960). The biopsies were done on an outpatient basis, all patients tolerating the procedure extremely well, a few experiencing pain at the operative site for several hours. Five of the patients had been receiving daily doses of neostigmine or mestinon, while one was receiving no medication. In no instance was the ordinary daily medication altered during the day of the biopsy. Control muscles were obtained during thoracotomy procedures on patients of both sexes and with no Imown muscle disease. The usual techniques were employed for intracellular recording with capillary glass microelectrodes (Fatt & Katz, 1951) and for iontophoretic micro-application of acetylcholine (del Castillo & Katz, 1955). A change in the composition of the external fluid was produced either by altering the bathing fluid or by superfusing the end-plate region with solutions delivered from a pipette, as described by Nastuk (1959). The basic composition of the bathing fluid was that used by * John and Mary R. Markle Scholar in Medical Science (Neurology), Present address: Division of Neurology, School of Medicine, University of Virginia, Charlottesville, Virginia.
NEUROMUSCULAR JUNCTION IN MYASTHENIA GRA VIS 337 Liley (1956a). Alterations in potassium concentration were accompanied by an iso-molar change (of opposite sign) in sodium concentration. Indirect stimulation was accomplished by stimulating fine nerve branches through a micro-pipette filled with 3M-NaCl solution. The duration of the stimulating impulse was l0fisec..~~~~~~~~~i AI Fig. 1. The end-plate region of a single fibre from a myasthenic patient was localized (A) by the recording of an e.p.p. with a fast rising phase, or (B) by the recording of a transient membrane depolarization in response to iontophoretically applied ACh. The current through the pipette appears in the lower tracing and is equivalent to a total charge of 2 x 10-10 C. A.C. artifact 50 c/s. Voltage calibration, 1 mv. RESULTS The resting membrane potential of single muscle fibres was 70-90 MV, which is similar to that of normal muscle (Elmqvist et al. 1960). In the muscles obtained from the six myasthenic patients 97 end-plate reg'ions were located by direct observation of fine nerve branches and either by the recording of an end-plate potential (e.p.p.) with a fast rising phase (Fig. IlA), or by the recording, as shown in Fig. 1 B, of membrane depolarization in response to iontophoretic micro-application of acetylcholine (ACh). The total length of each of the ACh-sensitive areas was 0* l-0*2 mm. 22-2
~~~~~~~~~~~~~~~~~~1 mv 338 0. DAHLBACK AND OTHERS In the great majority of the fibres from myasthenic patients spontaneous prejunctional activity, recorded as miniature end-plate potentials (m.e.p.p.s), was markedly decreased in frequency, or completely absent. In but 3 fibres from one patient were m.e.p.p.s of about normal frequency (0*2/sec) recorded (Fig. 2). In the remaining fibres only occasional m.e.p.p.s were observed (1-2/3-5 min). The amplitude and the time course of the m.e.p.p.s were similar to those of normal muscle (Fig. 2). ii~~~~~~~~~~~~~~~~~~~~~~~i Dw - - _ U 20 n sec 2 sec Fig. 2. Spontaneous m.e.p.p.s recorded in a muscle obtained from a patient with myasthenia gravis. As demonstrated by Liley (1956b), an increase in the potassium concentration of the bathing fluid markedly increases the frequency of m.e.p.p.s. In the rat diaphragm the frequency of m.e.p.p.s increases about 1000 times in response to a 30 mm potassium solution and this change persists for at least 20 min. The amplitude of the m.e.p.p.s is reduced in proportion to the potassium-induced fall in the resting membrane potential of the muscle fibre (Liley, 1956b). In normal human intercostal muscles a change in the potassium concentration from 5 to 30 mm invariably produced a long-lasting hundred- to thousandfold increase in the discharge frequency of m.e.p.p.s (Fig. 3 A, B). Just as invariably, on the other hand, the application of a similar potassium concentration to muscles from myasthenic patients produced no increase in the frequency of m.e.p.p.s (Fig. 3C, D). Even after long periods of immersion (15-30 min) in solutions containing potassium in concentrations varying from 10 to 40 mm, no increase in the frequency of m.e.p.p.s was observed. The difference between normal muscles and those obtained from myasthenic patients was particularly well demonstrated when, instead of changing the bathing fluid, the neuromuscular junction was superfused by a solution containing 30 mm potassium. Immediately
NEUROMUSCULAR JUNCTION IN MYASTHENIA GRA VIS 339 following the potassium application in the normal muscle, there was a marked increase in the frequency of m.e.p.p.s, but no such change was observed in muscles from patients with myasthenia gravis (Fig. 4). An increase of the external calcium-ion concentration to twice normal did not alter the response of myasthenic muscles to potassium. 2 sec Fig. 3. The frequency of m.e.p.p.s in the presence of a solution containing 5 mm potassium (records A, C) and a solution containing 30 mm potassium (records B, D). Records A and B are from a normal muscle and C and D from a muscle of a myasthenic patient. Time marker, record B, 20 msec. With potassium in concentrations of 20-30 mm the resting membrane potential of muscle fibres was reduced to 45-65 mv in both normal muscles and those from myasthenic patients. With the pipette superfusion technique the resting membrane potential was higher. The transmitter release produced by nerve stimulation can be studied by determinations of the amplitude of e.p.p.s during and following periods of repetitive nerve stimulation. Therefore, the e.p.p. was recorded by intracellular electrodes in muscles from normal and myasthenic patients. In order to block neuromuscular transmission in normal muscles, tubo-
340 0. DAHLBACK AND OTHERS curarine was used (0*2-0*3 mg/100 ml.). In myasthenic muscles a short period of high-frequency nerve stimulation produced a lasting block of transmission to the majority of the fibres. In normal muscles a brief period (2-3 sec) of repetitive nerve stimulation at a frequency of 100/sec or more, produced, after a short delay, a marked increase in the amplitude of the e.p.p., which increase lasted for about 1 min. In myasthenic muscles there was either no post-tetanic facilitation of the e.p.p., or only slight and short-lasting facilitation (Fig. 5). The amplitude of successive subthreshold e.p.p.s in a muscle from a myasthenic patient fluctuated at random without a progressive decline. In normal curarized muscle a similar stimulation frequency rapidly reduced the e.p.p. to a low and relatively stable value (Fig. 6). -~ ~ i Fig. 4. Wrhen the end-plate region of a fibre is superfused from a pipette with a solution containing 30 mm potassium, a marked increase in the m.e.p.p. frequency occurs in normal muscle (A). Record B is from the same fibre after 60 min of continuous superfusion. No m.e.p.p. discharge is observed in a mya-sthenic muscle in response to a similar application of potassium (C). The m.e.p.p.s was recorded with an a.c. amplifier while the d.c. potential of the fibre is shown in the lower tracing. Onset of superfusion is marked by interruption in the tracings. Time marker, 10 sec. Voltage calibration for upper tracings: records A and B, 1 mv; record C, 0-5 mv; for lower tracings in all records, 10 mv. DISCUSSION The recording of m.e.p.p.s with normal amplitude and time course indicates that the chemical sensitivity of the post-junctional membrane in muscles from myasthenic patients is similar to that in normal healthy subjects. Furthermore, end-plate 'spots' with a high sensitivity to ACh were observed in myasthenic muscles (Fig. 1 B). These findings make it unlikely that the neuromuscular defect is of post-junctional origin.
NEUROMUSCULAR JUNCTION IN MYASTHENIA GRA VIS 341 In muscles from patients with myasthenia gravis the low spontaneous m.e.p.p. frequency, and the lack of a frequency increase in response to potassium, suggest a pre-junctional deficiency affecting transmitter release. This possibility is supported by the absence of significant posttetanic facilitation of the e.p.p. in such muscles. As was shown by del Castillo & Katz (1954) and Liley (1956b), potentiation of the e.p.p. in response to nerve stimulation involves a recruitment of transmitter Fig. 5. E.p.p.s before (left-hand records), during (middle records) and 10 sec after (right-hand records) a period ofrepetitive nerve stimulation at about 100/sec. The upper records are from a curarized muscle of a normal patient and the lower ones from a non-curarized muscle of a myasthenic patient. Note that in the normal patient post-tetanic facilitation of the e.p.p. is responsible for a movement artifact. Voltage calibration: upper records, 1 mv; lower records, 0-5 mv. release. The amplitude of successive e.p.p.s in the myasthenic preparation fluctuated at random, and depression of the response to high-frequency stimulation failed to occur when the e.p.p. was already reduced to a subthreshold level by a preliminary period of nerve stimulation. Del Castillo & Katz (1954) demonstrated that such behaviour characterizes e.p.p.s in the presence of high magnesium- and low calcium-ion concentrations, i.e. when the quantal ACh content of transmitter release is reduced to a low value.
342 0. DAHLBACK AND OTHERS Thus, the results obtained from the present studies of spontaneous transmitter release, as well as of the e.p.p., strongly indicate that there exists in myasthenia gravis a deficiency affecting either transmitter formation or the transmitter release mechanism. Desmedt (1957, 1958) has reached a similar conclusion after studying post-tetanic effects with the electromyogram and isometric myogram in myasthenic patients. 11 II 'I 11 Fig. 6. E.p.p.s in response to repetitive nerve volleys at 100/sec. The upper record is from a normal curarized muscle and the lower one from a non-curarized muscle obtained from a patient with myasthenia gravis. Voltage calibration, 1 mv. None of the myasthenic patients in our study had clinical signs or symptoms indicative of involvement of the intercostal muscles (nor of the diaphragm). Yet the pathological-physiological changes described in the present experiments were present in the intercostal muscles of all patients. Therefore it is likely that the disease involves all the skeletal muscles, but is clinically evident as weakness only in those most severely affected.
NEUROMUSCULAR JUNCTION IN MYASTHENIA GRAVIS 343 SUMMARY 1. Some of the electrophysiological properties of the neuromuscular junction have been studied by the use of intracellular electrodes in isolated intercostal muscles obtained from patients with myasthenia gravis and from those with no known neuromuscular disorder. 2. Spontaneous pre-junctional activity, recorded as miniature endplate potentials (m.e.p.p.s) was markedly decreased in frequency in muscles from myasthenic patients. The application of a solution containing 10-40 mm potassium produced no increase in the frequency of m.e.p.p.s in myasthenic muscles, whereas in normal muscle it invariably caused a hundred- to thousandfold increase in the discharge frequency of m.e.p.p.s. 3. In normal muscle a short period of high-frequency nerve stimulation produced a marked post-tetanic facilitation of the amplitude of the endplate potential (e.p.p.). In myasthenic muscle no post-tetanic facilitation, or only slight and short-lasting facilitation, was observed. During tetanic nerve stimulation the amplitude of successive subthreshold e.p.p.s in myasthenic muscles fluctuated at random without a progressive decline. In normal curarized muscle a similar stimulation frequency rapidly reduced the e.p.p. to a low and stable value. 4. It was concluded that the results obtained from the present studies of spontaneous transmitter release and of the e.p.p. strongly indicate that there exists in myasthenia gravis a prejunctional deficiency affecting either transmitter formation or the transmitter release mechanism. We are greatly indebted to Professor T. Broman and the Department of Neurology, Sahlgrenska sjukhuset, Gothenburg, for their assistance in obtaining patients for this study. This investigation was aided by United States Public Health Service research grant B-2646 from the National Institute of Neurological Diseases and Blindness, by the Muscular Dystrophy Associations of America, Inc., and by the Air Research and Development Command, United States Air Force, through its European Office. REFERENCES DEL CASTILLO, J. & KATZ, B. (1954). Statistical factors involved in neuromuscular facilitation and depression. J. Physiol. 124, 574-585. DEL CASTILLO, J. & KATZ, B. (1955). On the localization of acetylcholine receptors. J. Physiol. 128, 157-181. DESMEDT, J. E. (1957). Nature of the defect of neuromuscular transmission in myasthenic patients: 'post-tetanic exhaustion'. Nature, Lond., 179, 156-157. DESMEDT, J. E. (1958). Myasthenic-like features of neuromuscular transmission after administration of an inhibitor of acetylcholine synthesis. Nature, Lond., 182, 1673-1674. ELMQVIST, D., JOHNS, T. R. & THESLEFF, S. (1960). A study of some electrophysiological properties of human intercostal muscle. J. Phy8iol. (In the press.). FATrT, P. & KATZ, B. (1951). Analysis of the end-plate potential recorded with an intracellular electrode. J. Physiol. 115, 320-370. LILEy, A. W. (1956a). An investigation of spontaneous activity at the neuromuscular junction of the rat. J. Physiol. 132, 650-666. LILEY, A. W. (1956b). The effects of presynaptic polarization on the spontaneous activity at the mammalian neuromuscular junction. J. Physiol. 134, 427-443. NASTUK, W. L. (1959). Some ionic factors that influence the action of acetvlcholine at the muscle end-plate membrane. Ann. N.Y. Acad. Sci. 81, 317-327.