An electrophysiological and morphological study of the neuromuscular junction in patients with myasthenia gravis

EXPERIMENTAL

NEUROLOGY

536-563 (1976)

51,

An Electrophysiological and Morphological Study of the Neuromuscular Junction in Patients with Myasthenia Gravis E.X.

ALBUQUERQUE,

Departments

of

J.E. RASH, R.F. MAYER,AND Pharmacology

and

Experimcv~tal

Surgery, University of Maryland, Baltimore, Received

January 12,1976;

Maryland revision

J.R. SATTERFIELD~

Therafieutics, Neurology of Medicine,

and

School 21201

received January 28,1976

Three types of neuromuscular junction were found in the surface fibers of internal and external intercostal muscles from patients with myasthenia gravis. One group (about 25% of the fibers) responded to nerve stimulation with an endplate potential (EPP) large enough to trigger an action potential and was associated with relatively mild morphological alterations in the postjunctional membrane. A second group had EPPs of markedly reduced amplitude and was associated with grossly altered postjunctional membrane structure and slight to moderate nerve degeneration. The spontaneous miniature endplate potentials (MEPPs) found in this group were markedly reduced in amplitude and frequency. In the third group of surface fibers neither EPPs nor spontaneous MEPPs were recorded at the endplate region. These endplates exhibited gross alteration of the cleft and folds and their nerves were absent or degenerating. Microiontophoretic application of acetylcholine (ACh) at the endplate region of all three groups of surface fibers disclosed low values of ACh sensitivity at the endplate region. No increase in extrajunctional sensitivity to ACh was detected in the myasth’enic muscles. There was no significant difference in membrane potential between normal and myasthenic muscles. Electron microscopic analysis of fibers from each grou,p revealed acontinuumof alterations in thmesynaptic folds and the sequential destruction of nerve terminals. The appearance of “fuzzycoated” vesicular remnants of degenerating folds and the invasion of the 1 Drs. Albuquerque and Rash ‘are in the D’epartment of Pharmacology and Experimental Therapeutics. Dr. Mayer is in the Department of Neurology and Dr. Satterfield is in the Department of Surgery. Supported by USPHS Grant NS-12063 and the Muscular Dystrophy Associations of America. Dr. Mayer is supported in part by USPHS Grants NS-06779-08 and NS-5077-21 and by the Potomac Valley Chapter of the Myasthenia Gravis Foundation. The authors are ind’ebted to Ms. Mabel A. Zelle for secretarial and technical assistance. 536 Copyright All rights

0 1976 by Academic Press, of reproduction in any form

Inc. reserved.

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endplate region by a variety of leucocytes suggest the occurrence of an immune response. Thus, in myasthenia gravis, alterations of the synaptic folds are associated with a decrease in ACh receptor density, thereby accounting for the marked reduction in the membrane response to endogenous or microiontophoretically-applied ACh at the endplate region.

INTRODUCTION Myasthenia gravis, a disease characterized predominantly by muscle weakness which usually becomes more evident upon repetitive motor activity, is currently thought to arise from a defect involving the endplate region. Early electrophysiological studies (13) demonstrated a decrease in the amplitude of both miniature endplate potentials (MEPPs) and endplate potentials (EPPs) and were interpreted as suggesting a decrease in the amount of acetylcholine (ACh) contained in each quanta. Because this proposed defect could account for the observed blockade of neuromuscular transmission upon repetitive stimulation (13)) it was widely assumed that myasthenia gravis could be attributed to a presynaptic defect, presumably of the motor nerve terminal alone. Recent studies have revealed that immunological factors may be important in the development of the disease process (25, 29, 30). S everal investigators subsequently compared the postsynaptic membranes of normal and myasthenic patients and observed that the density of cholinergic receptors at the endplate region is markedly reduced in the muscles of patients with myasthenia gravis (5, 17). Other investigators have observed gross alteration in the postsynaptic junctional folds (16, 28) and have speculated on the possible adverse effects attributable to the increased distances between the presynaptic and postsynaptic membranes at the endplate region. Because the distance between the presynaptic and postjunctional membranes in amphibian and mammalian neuromuscular junctions is maintained at about 500 to 500 K (7-9, 12, 22), the architecture and composition of the normal synaptic cleft may be critical for the successful interaction of sufficient numbers of transmitter molecules and receptor sites activated during the initiation of normal muscle EPPs, thereby insuring that the transient amplitude is of the appropriate magnitude to trigger action potentials (1). Furthermore, the studies on density and distribution of putative ACh receptors at the postjunctional membrane by Fertuck and Salpeter (19, 20)) Albuquerque et al. (1)) and Rash and Ellisman (26) have revealed that in normal ~mznz~~zaZian muscles the largest density of ACh receptors is at the top of the synaptic folds. At those regions, large numbers of receptors are observed (about 20,000 to 30,000 a-bungarotoxin binding sites/pm” (1, 6, 20) or the equivalent of 1800 to 2000 putative ACh receptor octomeric complexes/& (12, 26, 27). The density of ACh receptors at the top of the folds appears to be constant among mammals,

538

ALBUQUERQUE

ET

AL.

implying that this density combined with the rigidly controlled diffusion distance, rather than the total size of the endplate, is critical for the generation of synaptic potentials ( 1, 12). The purpose of the present study is to investigate in more detail whether alterations in synaptic transmission in myasthenic muscles result from a decrease in transmitter release, a decrease in density of ACh receptors at the postjunctional membranes, an alteration in cleft morphology, or a combination of all these factors. In the present investigation we have used microiontophoretic application of ACh to determine the chemosensitive properties of the normal and myasthenic muscles, followed by systematic electron microscopic analysis of fibers from the same muscle bundles and from additional muscles not otherwise tested. Electrophysiological analysis using accurate placement of the micropipette filled with ACh within the endplate region, and involving detailed mapping of the ACh sensitivity of individual fibers from endplate to tendon are correlated with specific morphological changes in fold morphology and cleft composition, thereby clarifying several aspects of the nature of the postsynaptic defect involved in myasthenia gravis. Furthermore, our data provide electron micrographic evidence compatible with an immunologically mediated destruction of junctional fold and junctional cleft constituents. Thus, it is suggested that the degenerative process of myasthenia gravis has a complex nature involving a decrease in ACh receptor density, alterations of the postjunctional juxtaneural region, and, to a much lesser extent, degeneration of the presynaptic nerve terminal. MATERIALS

AND

METHODS

Patients. Five patients with myasthenia gravis (myasthenic) and six control patients were studied. The patients with myasthenia gravis were female, age 12 to 52 yr (Table 1). Duration of symptoms ranged from 3 months to 4 yr. All had generalized myasthenia gravis at the time of study involving bulbar and respiratory muscles. Two patients had been receiving anticholinesterase (AChE) drugs and alternate-day steroids for 6 to 20 months. The third patient had received anticholinesterase drugs for only a few weeks, but this treatment was stopped 6 weeks prior to study. The fourth and fifth patients had received only small amounts of pyridostigmine for 6 weeks. All had thymectomies performed because of progressive weakness. In one, the size of the thymus was found to be enlarged by radiological examination during the period of observation and was subsequently found to be a malignant thymoma with multiple small metastases. At the time of surgery, a full-thickness intercostal muscle (sixth intercostal space, anterior to midaxillary line) biopsy was obtained from each patient using standardized anesthesia and surgical technique. Similar pieces of intercostal

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ET AL.

muscle were removed from six patients who demonstrated no clinical evidence of neuromuscular disease. These patients (four males and two females, age 29 to 62 yr) were undergoing thoracotomy because of cardiovascular or pulmonary impairment. Two small samples of the intercostal muscle were removed, one immediately frozen in isopentane and dry ice for histochemical study, the other fixed in ice-cold 2.5% glutaraldehyde for electron microscopic study. A larger sample of muscle was removed and immediately placed in a large volume of highly oxygenated Krebs-Ringer solution as described previously (2, 14) ; special care was taken during the surgery in removing the intercostal muscles to allow dissection of bundles of the external and internal intercostal muscles. The muscles were then microdissected into small bundles for electrophysiological and electron microscopic analysis. In one case, small bundles were prepared for study using Nomarski interference optics. Under continuous flow of physiological solution the muscles were microdissected to remove all possible connective tissue, separated into bundles, and placed in the recording chamber kept at room temperature or warmed to 34°C. It should be noted that for Nomarski studies, one bundle each of intercostal muscles from normal and myasthenic patients was also treated with collagenase to remove additional connective tissue, thus allowing better separation into bundles. The muscles treated with collagenase showed no significant electrophysiological differences from control muscles (2). After isolation of bundles, the muscles were put into the recording chamber and left to equilibrate for at least 30 min. Electrophysiological Techniques. Conventional microelectrode techniques were used for intracellular stimulation and recording from intercostal muscles (3). In the studies using microiontophoretic application of ACh, high-resistance pipettes (about 150 * 40 Ma) filled with 2.5 M ACh were used at the junctional and extrajunctional regions of the normal and myasthenic muscles. The duration of the cathodal current pulse varied from 0.5 to 1.0 msec, and this current pulse was superimposed on a low constant anodal braking current. In several attempts to bring the ACh depolarization to similar levels as those obtained in the control condition, much larger charges were required with marked deformation of the current pulse. Under this circumstance, proper calculation of the total ACh sensitivity in mV/nC would be grossly in error. Moreover, in only a few successful encounters were we able to obtain ACh potentials with an amplitude greater than 5 mV in the myasthenic muscles lacking MEPPs or EPPs (see Fig. 4C3, 4). Thus, the duration of the ACh pulse was maintained constant in both normal and myasthenic muscles. When an increase in charge applied to these high-resistance pipettes was required (as occurred in the case of the myasthenic endplate region), precautions were taken to

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avoid capacitance leakage. Extrajunctional ACh sensitivity values were not corrected for membrane potential since there was very little variance from fiber to fiber. For studies using Nomarski interference optics, very small bundles (usually containing two or three layers of muscle fibers) were dissected. The position of the recording electrode at the endplate was considered focal when the rise time of the spontaneous MEPPs was about 1 msec. In preparations where no transmitter release was observed, the nerve twig was retraced and the apparent endplate region localized and mapped. For nerve stimulation either special double platinum or small suction electrodes were used. For the studies involving action potentials, the technique was as described previously (4). Electron Microscope Techniques. All samplesof the intercostal muscle were fixed for at least 1 hr in 2.5% glutaraldehyde dissolved in Tyrode’s solution or 0.15 M Sorenson’s phosphate buffer (25). Endplate regions were visualized by staining for 1 hr in buffered glutaraldehycle solution plus 0.01% acetylthiocholine iodide, 0.002 M CuS04, 0.01 M sodium citrate (pH 7.0). It should be noted that after anticholinesterase treatment, neuromuscular junctions stained very slowly, greatly increasing the difficulty in obtaining samples for electron microscopy. Portions of fibers containing the neuromuscular junctions were rinsed in 0.15 M phosphate buffer (pH 7.2), postfixed in buffered 1% 0~0~ or 0~0~ + 0.001 M K3Fe( CN)o, rinsed, and poststained in aqueous or methanolic uranyl acetate. Thin sections were stained with lead citrate and photographed in a Siemens Elmskop 101 operated at 80 kV. [For further details of these procedures, see (12, 18, 26) 1. RESULTS Neuronmscztlar Transmission in Normal and Myasthenic External and Internal Intercosfal &Jllsrles. In fibers from internal and external inter-

costal muscles of normal human subjects, indirect stimulation produced EPPs large enough to elicit an action potential in all fibers (Fig. lAl3). Indirect stimulation of the same muscles from myasthenic patients, however, resulted in EPPs large enough to trigger spike activity in only about 25% of the surface fibers tested (Fig. lB, Cl). Furthermore, when the recording microelectrode in the myasthenic fibers was not dislodged after the first action potential was generated, repetitive indirect stimulation at the rate of 10 Hz resulted in a decrease of the endplate potential to such low amplitude that no action potentials were generated. In fact, by the 30th to 50th potential, EPPs with amplitudes as low as 2 mV were observed. The majority of the endplate regions had subthreshold EPPs which were decremental in amplitude if stimuli were applied at a rate of

542

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ET AL.

FIG. 1. Response of the postjunctional membrane at the endplate region of normal and myasthenic, internal and external intercostal muscles. Al-3: action potentials elicited in three different surface fibers of normal internal (Al, 2) and external of three different (A3) intercostal muscles by indirect stimulation. El, 2: response surface fibers of an internal intercostal muscle of a myasthenic patient. In Bl, nerve stimulation elicited an EPP large enough to trigger an action potential. In B2, the small, subthreshold EPP was recorded, and in B3, neither an action potential nor an EPP could be obtained. Cl-3 refers to the response to nerve stimulation of three different surface fibers of the external intercostal muscle. In Cl, an action potential was obtained. In the same muscle, a second fiber showed an EPP of about 1 mV (C2), and in C3 another fiber responded to indirect stimulation with an EPP of only about 0.25 mV. Stimulation was at a rate of 1 Hz. The calibration for the action potential is shown in Al, the calibration for the EPP in B2, and these calibrations also refer to other traces.

> 10 Hz (Fig. lB, C2). Other fibers in the myasthenic muscles had no observable transmitter release, that is, one could elicit neither an EPP (Fig. lB3) nor recordable evoked transmitter release. Spontaneous transmitter release, if present, then, was below the noise level of the recording system. The resting membrane potential recorded in surface fibers of the internal and external intercostal musclesof nonmyasthenic patients was -79 * 1.10

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TABLE Resting

Membrane

MYASTHENIA

2

Potential and MEPP Amplitude Muscles of Normal and Myasthenic External Normal

Resting Potential

membrane

-79

543

GRAVIS

zk l.lOh (523)c

and Frequency Patients

in Intercostal

intercostal Myasthenic” -77

* 2.6s (293)

bV) MEPP Amplitude bv)

0.80 f 0.20 (211/360)”

0.32 zk 0.059~ (205/511)

MEPP Frequency (x-l)

0.33 zk 0.081 (211/1360)

0.22 f 0.011e (163/571)

Internal Normal Resting Potential (mV)

membrane

-81

zk 1.30 (524)

intercostal Myasthenic -79

It 2.07f (311)

MEPP Amplitude (mW

0.90 f 0.031 (202/550)

0.37 zt 0.072’ (193/400)

MEPP Frequency (set-I)

0.41 zt 0.072 (273/1109)

0.24 & 0.011” (193/462)

a Resting membrane potentials of mynsthenic muscles were recorded from all three groups of surface fibers, i.e., the group that responded to nerve stimulation with an EPP large enough to trigger an action potential, the group which had a markedly reduced EPP and reduced MEPP amplitudes, and the third group where neither EPPs nor spontaneous MEPPs were recorded. b Mean =k SEM. c Number of fibers studied. d Number of fibers/number of potentials counted. In 2.5 to 3O’r, of the myasthenic muscle fibers studied (both external and internal intercostal) MEPPs were not found. Number of fibers (myasthenic muscles) refers only to those fibers where MEPPs were recorded. e Difference from normal muscle is statistically significant (P < 0.001). ’ Difference from normal muscle is not statistically significant (P > 0.05).

mV and -81 -+ 1.30 mV, respectively (Table 2). Although a slight decrease in membrane potential was observed between normal and myasthenic muscles, the difference was found not to be significant (Table 2).

544

ALBUQUERQUE

ET

AL.

The spontaneous MEPP frequency recorded in the normal external intercostal muscles was slightly (but not significantly) lower than that in the internal intercostal muscles (Table 2)) whereas the spontaneous MEPP frequency in both internal and external intercostal muscles of myasthenic patients was significantly lower than that of the normal muscles (Table 1). The mean distribution of MEPP amplitudes in normal and myasthenic patients (Fig. 2A) showed a typical bell shape slightly skewed to the right. In normal fibers, the spontaneous MEPP amplitudes followed a normal distribution with more skewing to the right side of the median, with spontaneous MEPPs as high as 3 to 4 mV. The mean amplitude of

1 o

1 10

05 mepp

i //i-f/-l20 30

I5 AMPLITUDE

40

CmV)

FIG. 2. Distribution of mean amplitudes of the spontaneous MEPPs obtained from normal (A) and myasthenic (B) patients. The broken line refers to the amplitudes of the MEPPs in the internal intercostal muscles, and the solid line, the amplitudes of MEPPs in the external intercostal muscles. Since only slight variation in membrane potential was found, no correction of the MEPP amplitudes was made for membrane depolarization.

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the potentials (as shown in Table 1 and Fig. 2A) was not significantly different in the external and internal intercostal muscles. In contrast, only one out of 150 endplate regions studied in the myasthenic muscles revealed even one or two MEPPs with amplitudes as high as 2 mV. In the in general, then, the amplitude of the MEPPs was myas,thenic muscles, markedly decreased (to about 30 to 40% of normal), the mean being 0.34 mV for the external and 0.33 mV for the internal intercostal muscles (Table 1 and Fig. 2B). In fibers where no spontaneous MEPPs were recorded, stimulation of the nerve occasionally elicited EPPs of extremely low amplitudes, usually about 0.25 to 1 mV (Fig. lB2, 3). Furthermore, in the large number of myasthenic fibers where no spontaneous MEPPs or evoked EPPs could be elicited, neither treatment of the preparation with neostigmine (2 X 10-O g/ml) nor an initial treatment with diisopropylfluorophosphate (1.0 X 10m3 M) followed by wash for 60 min to remove excess drug would allow MEPPs or EPPs to be recorded (Albuquerque, Garrison, Mayer and Satterfield, unpublished observation). Thus, treatment with neostigmine appears not to increase the ability of grossly affected TABLE ACh

ACh

Normal patient no. External 2356 1975 2727 1830 2697 2021

Myasthenic patient no.

External

intercostal

fibersc

“B”

“A” 1

932 873 1023 975

f f xk +

431 210 198 203

sensitivity

of Intercostal Patients

dz + f f f f

(31) (47) (36) (15)

343c 476 333 411 443 397

Internal

(28)b (15) (22) (18) (18) (10)

397 323 401 514

2733 2531 2630 1983 2211 1930

intercostal f zk f f + It

Internal libersd

f f f f

Muscles

(mV/nC)

intercostal

1 2 3 4 5 6

2 3 5

3

Sensitivity at Endplate Regions of Normal and Myasthenic

189 97 121 161

(29) (30) (52) (16)

“A” 753 933 876 915

fibers’ f f zk f

210 310 119 123

176 217 233 401 311 199

(29) (18) (18) (17) (15) (14)

intercostal “B”

(28) (60) (39) (15)

397 583 433 486

fiber& zt 77 (29) f 102 (27) f 301 (36) i 110 (15)

a Mean f SD. 5 Numbers in parentheses represent the number of libers studied. c Group “A” fibers are from muscles in myasthenic patients in which MEPPS present. d Group “B” fibers refer to muscles where no EPPs or MEPPs were recorded.

were

546

ALBUQUERQUE

ET AL.

FIG. 3. The response to microiontophoretic application of ACh at the endplate region of surface fibers of the internal intercostal muscles of normal (A) and myasthenic (B and C) patients. Al-4 : typical recordings from four different endplate regions of normal patients; B1-4: of myasthenic patients where spontaneous MEPPs and EPPs were recorded; and Cl-4: of myasthenic patients where no spontaneous MEPPs or EPPs were recorded. Horizontal calibrations are time in msec: A-10, B-20, C-30 msec. The vertical upper bar is the calibration of the amplitude of the transient depolarization produced by ACh: A-5, B-4, C-Z mV. The lower vertical bar is 2 X lOWEA. Duration of the ACh pulse was 0.5 msec. Membrane potentials : Al-4 : -78, -76, -81, -80 mV; B1-4 : -74, -76, -80, -81 mV; Cl-4: -78, -75, -76, -79 mV.

fibers to elicit action potentials, but may serve only to increase the probability of firing in partially degenerated fibers ( 15). The

ACh

Sensitivity

of Norvvlal

and

Myasthenic

Intercostal

Muscles.

The mean values for junctional ACh sensitivity of the surface fibers of nortnal internal and external intercostal muscles (see Table 3) varied from about 1500 mV/nC to 3000 mV/nC. I n one case a value of 3130 mV/nC was found on a surface fiber of an external intercostal muscle fiber. Examination of muscles from myasthenic patients, however, consistently revealed significantly reduced ACh sensitivities (Table 3). In the least

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affected fibers, those where spontaneous MEPPs could be observed and where nerve stimulation elicited an EPP large enough to trigger an action potential, values for ACh sensitivity of about 650 to 1200 mV/nC were obtained. These values were significantly lower than those obtained in normal muscles. However, in those endplates without observable spontaneous transmitter release or detectable nerve-elicited EPPs, sensitivities to microiontophoretically applied ACh as low as 250 mV/nC were measured, indicating that these endplates did not have high enough density of activatable receptors to allow sufficient interaction with ACh. The rise time of the ACh potentials recorded at the endplate region of the surface fibers of normal internal and external intercostal muscles was from 1.2 to 6.0 msec (Fig. 3), whereas, in surface fibers of the myasthenic muscles, the rise time varied from 2.8 to 22 msec (Fig. 4). Furthermore, the slowest rise times in the myasthenic fibers were obtained from those endplates where neither spontaneous MEPPs nor EPPs could be elicited. In those muscles where no transmission was observed, the values of extrajunctional ACh sensitivity were similar to those obtained in muscles where transmission was present (Table 4). In addition, those muscle fibers did not have tetrodotoxin-resistant action potentials, which are commonly observed in chronically denervated extensor digitorum longus muscles of the rat (4). After ACh sensitivity was measured in virtually all surface fibers of a given muscle bundle, they were subjected to serial thin-sectioning for electron microscopic examination. Fibers from bundles where EPPs or MEPPs were recorded as well as those demonstrating no spontaneous or evoked EPPs were also examined. Ultrastructure. Following electrophysiological examination, muscle bundles were fixed by immersion in ice-cold phosphate buffered glutaraldehyde and prepared for routine transmission electron microscopy and for freeze-fracture analysis. Additional tissues from the same patients and from patients without myasthenia were fixed by immersion immediately after biopsy. An endplate from a nonmyasthenic patient exhibiting apparently normal morphology is illustrated in Figs. 5 and 6. At low magnifications (Fig. 5b), the endplate region is discerned as a 30 pm region, containing (in this plane of section) five terminal swellings of the motor nerve (arrows). Cytoplasmic extensions of the overlaying Schwann cell are closely applied to each nerve swelling. A subsequent nonadjacent serial section (Fig. SC) helps to reveal the interconnecting complexity of the convoluted junctional folds (see arrows in Figs. Sb, c). Regular spacing and relatively uniform depth of the junctional folds are clearly evident. At higher magnifications (Fig. 6), a nerve terminal expansion is seen filled with 500-to-600-A synaptic vesicles, a few mitochondria, and a moderate

548

ALBUQUERQUE

FIG. 4. The description of this figure fibers are from the external intercostal -74, -78 mV; B1-4: -78, -77, -75, mV.

ET AL.

is as given muscles. -81, -75,

for Fig. 3, except that the surface Membrane potentials : Al-4: -77, mV; ClL4: -74, -79, -78, -75

amount of smooth membrane profiles interpretable as the cisternae associated with membrane recycling (22) or as smooth endoplasmic reticulum. The width of the primary synaptic cleft is seen to be rigidly maintained at 700 to 900 A, with a uniform basal lamina (*) interposed between nerve and muscle and between successive junctional folds. The clefts are virtually devoid of other structures or material, the small amount of residue present possibly reflecting normal metabolic turnover of junctional material. Characteristically, the membranes of the crests of the junctional folds are of greater thickness and electron density than the membranes of other portions of the folds (Fig. 6), presumably reflecting the high concentration of membrane integral substances associated with the juxtaneural distribution of ACh receptors (1, 19, 26). In muscles from five patients with myasthenia gravis, a continuum of degenerative changes was observed in the approximately 20 randomly and serially sectioned endplates. At low magnification (Fig. 7a), most of the

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TABLE Extrajunctional Normal Distance endplate

from (rm)

ACh Sensitivity and Myasthenic

2150 8.5 5 0 0.38 0

f f f f f f

3018 (19)D 23 (18) 1 (18) 0 (17) 0.24 (15) 0 (14) Internal

0 100-200 500 1000-1300 1500-1700 2000-3000

2750 121 3.5 0.04 1.0 0

f f f f f &

317 (26) 49 (21) 12 (19) 0.01 (19) 0.7 (18) 0 (17)

549

GRAVIS

4 in Intercostal Muscles Patients (mV/nC) M Y asthenic

Normal

External 0 100-200 500 1000-1300 1500-1700 2000-3000

MYASTIIENIA

“A”c

of

M Y asthenic

“B’ld

intercostal 1101

f

443 (16) 7 (19) 0.01 (17) 0 (17) 0.02 (16)

750 146 23 0.55 0 0.01

zt f f * + f

233 (22) 83 (22) 7 (21) 0.01 (21) 0.6 (20) 0.03 (21)

391 171 33 1.45 0.01 0.09

f 178 (18) * 74 (17) l 11 (17) z!z 0.66 (17) f 0.01 (17) z!z 0.04 (17)

75 f 30 (11) 10 0.02 0 0.08

f f f f

321 (18) 73 (18) 10 (13) 0.17 (13) 0 (14) 0.01 (14)

intercostal 978 211 13 0.02 1.20 0.02

f f f f xt +

a Mean f SD. b Numbers in parentheses represent the number of fibers studied. c M Y asthenic “A”: myasthenic muscles where MEPPS were recorded. d Myasthenic “B” : myasthenic muscles where no MEPPs were recorded.

endplates from patient #2 were recognizably altered in a manner characteristic of myasthenia gravis (16). Beneath the apparently normal Schwann cell caps, small nerve terminals were associated with irregular and distorted profiles of the junctional folds. Other myofiber components (such as nuclei, mitochondria, sarcoplasmic reticulum, and T-system) appeared relatively well organized. In the subjunctional perinuclear region, however, a large amount of interwoven tubular membrane profiles and “myelin whorls” of unknown origin are consistently observed. At higher magnifications (Fig. 7b), the membranes of many of the crests of the folds are seen to have the same thickness and electron density as other cell membranes (arrow), with only scattered crests either of the characteristically increased density or closely approaching the nerve terminal. At very high magnification (Fig. 7c), a detached membrane profile, tentatively identified as a degenerating remnant of a junctional fold (cf. 16), is observed and is seen to possess an unusual SO-to-100-A layer of material inside the characteristic basal lamina (*). Often, especially in very small membrane remnants (Fig. 7d, e), this (or a similar) layer appears to be composed of numerous

550 _..._” I

ALBUQUERQUE . .._-

-..

- -....

..__-----.---

ET AL. ---

^.

_

t I

Y

FIG. Sa.-Motor endplate region obtained by surgical biopsy from a nonmyasthenic patient. The five terminal expansions of the nerve (arrows) are closely opposed by the regular infoldings of the neuromuscular junction. Portions of at least two Schwann cells overlie the endplate, extending and virtually surrounding one nerve expansion. b, c--Nonadjacent serial sections showing complex infoldings and interconnections of junctional folds (compare large arrows). Occasional apparently increased separation of folds (Sb, arrow) revealed to be plane of section artifact resulting from sectioning parallel to and between successive folds (5c, arrow). Cilium (C) of Schw’ann cell. a: X 5000; b and c. X 7500.

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FIG. 6. Higher magnification of inscribed area in Fig. 5~. Synaptic vesicles (SV) and normal complement of mitochondria in nerve terminal swelling. The basal lamina (*) or basement membrane matrix, interposed between nerve and muscle and successive junctional folds, serves to mark the remnants of junctional folds following fold degeneration (see Figs. 7 and 8). The juxtaneural distribution of ACh receptors is thought to contribute to the increased thickness and electron density of membranes as the tops of the folds compared with other cell membranes (see double arrow). at the tops of the folds compared with other cell membranes (see double arrow). x 55,000.

552

ALBUQUERQUE

ET AL.

endplate region from myasthenic patient #2. Junctional folds grossly FIG. 7a.-Motor altered, with junctional clefts widened and filled with globular and membranous material. Nerve terminal cytoplasm partially depleted of synaptic vesicles but containing abundant mitochondria. Schwann cell and muscle cytoplasm relatively normal. b.-Intermediate magnification micrograph of clefts and folds. Junctional fold membranes remaining near nerve terminal (arrow) are of same thickness and electron density as other cell membranes. The basal lamina (*) remains around intact f_qlds and the larger degenerating remnants of folds. c, d.-High magnification micro-

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periodic and aperiodic projections, some of which resemble the letters “H” or “Y” (Fig. 7d, arrowhead). This unusual layer is apparently attached or absorbed directly to dense material within the membrane bilayer. In size and shape, these attached elements resemble the attachment of IgG and IgM immunoglobulin labels to virus coats in negatively and positively stained material (21, 23, 24). Other very small vesicular structures resembling further stages in the degeneration of junctional fold membrane and cytoplasm are confined within the remaining clefts but are devoid of both the basal lamina coating and the unusual “fuzzy coats” (Fig. 7e, arrow). The denuded membrane remnants and the remaining globular material may contain elements from several sources, some membranous, others cytoplasmic, and still others of unknown origin. [For example, compare globular material to IgM gammaglobulin fraction as reported by Hoglund (23) 1. In normal neuromuscular junctions (Fig. 5) a characteristic 300-to-500A basement membrane matrix (*) separates the nerve and muscle and is observed coating and separating each junctional fold. This basal lamina remains to mark the original position of the degenerating and degenerated folds in myasthenia gravis (Fig. 8a) and appears to restrict the diffusion of membrane remnants. Micrographs are thus assembled (Figs. Sa, b) which may be interpreted as forming a degenerative sequence in junctional fold destruction. Furthermore, serial sections (Fig. 8b, c) reveal that the absence of junctional folds between basal lamina infoldings does not result from a plane of section artifact. These neuromuscular junction “ghosts,” thus, appear to represent the terminal stage of endplate destruction. Additional evidence for the occurrence of an immune response is provided by the relatively frequent images of various types of leucocytes observed in the vicinity of degenerating motor endplates, both in thin sections (Figs. 9a-b and 10a) and freeze-fracture (Fig. 9c). Several of these cells (Fig. 9a, c) appear identical to the basophilic granular leucocytes designated mast cells or mastocytes (31) and others (Fig. lOa, b) resemble macrophages (Fig. 6a, arrow), small lymphocytes, and monocytes. Whether these images represent evidence for an autoimmune response to specific neuromuscular antigens or represent evidence for phagocytic activity associated with postdegenerative “clean-up” remains to be established. graph of junctional fold remnants and globular cleft material. Larger membrane remnants (Fig. 7c) often reveal a 50-to-loo-A-wide layer adsorbed, apparently separated by approximately 40 A. In smaller remnants, the adsorbed layer appears to consist of discrete elements shaped like small 100-A butterflies (Fig. 7d, large arrowhead), each apparently attached to electron-dense membrane integral components (small arrowhead, Fig. 7c and d). Still other remnants (Fig. 7e) appear as globular material (arrow) resembling other IgM gamma-gl’oublin fractions (cf. 2.2). a: x 5000; b: x 28,000; c : X 120,000; d : X 136,000; e: X 120,000.

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FIG. S.-Partially degenerated junctional fold membrane (open arrow) still encased in basement membrane sheath (*>. b, c.-Nonconsecutive serial sections of junctional fold “ghosts” as delineated by basement membrane convolutions (*). Vesicular (arrows) and globular (arrowheads) remnants remain in area formerly occupied by endplate. a-c : X 27,000. Degenerative processes involving the nerve terminals, too, may be inferred from the electron micrographs. Because apparently normal junctional folds are always found with normal nerves whereas apparently

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FIG. 9a, b.-Conventional electron micrograph of granular leucocyte (9a) observed within 50 pm of the endplate region in myasthenic muscle. Unambiguously identified as mast cell by the presence of small granules containing characteristic “scrolls” (9b, arrows) (28). C.-The relative abundance of mast cells is reflected in the numerous images obtained in freeze-fracture replicas from samples of endplate regions. The surface of the mast cell granules (MG) is seen to be convoluted, apparently reflecting the contour of the included “scrolls”. The folded cell surface is visible at the upper right (arrow) and the nucleus (N) with its double nuclear membrane containing nuclear pores is visible at the bottom. a : X 17,000 ; b : X 40,000; c: x 28,000.

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FIG. lOa.-Endplate region with closely associated phagocytic. cell tentatively id entified as macrophage (arrow). b, C.-Degenerating endplate with unidentified cell in junctional cleft region. Cytoplasm filled with granular and filamentous material vesicles opposite a degenerating junctional fold. This cell ar Id a few 400 to 600-A m ay represent a partially degenerated nerve terminal or an invading leucocyte, but th ,e absence of serial sections ,prevents positive identification. a : X 13,000; b : X 6000; c: x 26,000.

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FIG. lla-d.-Degenerative changes in nerve terminal associated with junctional fold destruction. Obvi’ous changes include reduction in size of nerve terminal and conoomitant reduction in number of synaptic vesicles. Compare with Figs. 6a, 7a, lOc, and 8a-c. a: X 13,000; b : X 26,000; c : X 16,000; d : X 16,000.

normal nerves may accompany grossly altered folds, degeneration of folds appears to precede or at least accompany nerve degeneration (see Figs. lob, c and 1 la-d). In several instances, the area normally occupied by the terminal nerve swelling was seen to contain a portion of a cell filled with

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filamentous material. Insufficient serial sections were obtained to ascertain whether this cell was an aberrant, degenerating nerve cell or was perhaps a nucleated lymphocyte. As yet, we have been unable to identify unambiguously either nerve terminals or junctional folds in our freeze-fracture replicas of grossly affected myasthenic endplates. However, in light of the above data obtained from thin sections (Fig. 7b, c) supporting the possibility of active ACh receptor removal from the folds, we are currently reevaluating all replicas of myasthenic endplates, in the expectation that other markers can be obtained for identifying and characterizing affected neuromuscular junctions. DISCUSSION The present investigation shows that the postjunctional membrane at endplate regions of normal human external and internal intercostal muscles has a sensitivity to microiontophoretically applied ACh similar to that observed in other mammals (1, 2, 6) but that ACh sensitivity in endplates from patients with myasthenia gravis is significantly decreased. These findings conform the initial observations made by Albuquerque et al. [see Table 8, ref. (2) 1. The decrease in ACh sensitivity is clearly evident in all surface fibers of myasthentic muscles, even in those fibers in which neuromuscular transmission can be observed. The low ACh sensitivity obtained in myasthenic muscles may be attributed to the degeneration of the synaptic folds, and perhaps to blockade of the ACh receptor by specific immunoglobulins. The fact that the endplate region of a myasthenic muscle contains only 4.8 to 11.4 X lo6 ACh receptors in contrast to 38.9 x lo6 in normal muscles (17) provides further support to our findings. However, even with this overall reduction of the number of ACh receptors, one should be able to obtain a higher ACh sensitivity than that which we have observed in the myasthenic muscles provided that a high ACh receptor density had been maintained in several “tops of the folds.” Under this condition, it is possible that a total decrease in packing of ACh receptors at the top of the folds had occurred in addition to the destruction of the synaptic fold as observed in the present studies. Our findings markedly differ from those described by other investigators (10, 13). For example, it was previously reported that the endplates of patients with myasthenia gravis have junctional ACh sensitivity similar to normal muscles (10). However, if the ACh sensitivities reported in that study are expressed in the conventional units (mV/nC), the values prove to be much smaller than those currently obtainable for normal human muscles or even for the myasthenic muscles. The discrepancy is most likely attributable to the technique of microiontophoresis used and to the use of

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low-resistance microelectrodes which result in unavoidable desensitization of the ACh receptor sites (11) ; consequently the data can no longer be accepted as reflecting either similarities or differences in normal vs. myasthenic motor endplates. Furthermore, the conclusion that there is a decrease in quanta1 size of the transmitter in myasthenia gravis patients (13) would be valid only if the geometry of the presynaptic and postjunctional membranes were unaltered and if the transmitter substance could reach areas of high receptor density. The present results indicate that the probability of interaction between the ACh released and those areas of the postjunctional membrane containing ACh receptors is markedly reduced, perhaps accounting for much ‘of the difficulty encountered by previous investigators (13) in attempting to evaluate the quanta1 size of the transmitter. Similarly, in fully 25 to 30% of the surface fibers examined in the five patients studied, our recording system was not able to reveal either the presence of MEPPs or nerve-stimulation-elicited EPPs. Subsequent electron microscopic analysis of the surface fibers in the bundles in which there was no detectable transmitter release disclosed a marked alteration of the postjunctional membrane. The postjunctional membranes were disrupted and the synaptic clefts were greatly and irregularly widened. Thus, the areas where the richest density of ACh receptors are located in normal tissues were often grossly deformed or completely absent in the myasthenic synaptic fold, and in these instances they were apparently replaced by coated membrane vesicles and deposits of small globular materials. Although the increase in the distance between the presynaptic and postsynaptic membranes and the marked degeneration of the junctional folds observed in myasthenic muscles certainly could account for the observed decrease in amplitude of the MEPPs, it is difficult to conceive that all synaptic folds in all endplates of those muscles are far away from the presynaptic nerve terminal. In fact, we found that in many fibers where MEPPs and low-ampltiude EPPs were recorded, many folds were at the normal distance from the presynaptic nerve terminal. If these folds had a normal density of ACh receptors, the distribution of MEPP amplitudes would be more skewed than that observed under normal conditions and such skewing was not seen in myasthenic muscles (see Fig. 2). In order for the bell-shape distribution of MEPP amplitudes to be similar in the normal and myasthenic intercostal muscles, there must be a relative decrease in the density of receptor sites, even at the top of those folds which had remained at the normal distance from the nerve terminal. Thus, electrophysiologic and electron microscopic evidence suggest that two alterations are occurring at the myasthenic endplate : (i) Gross degeneration of the folds with a large increase in the presynaptic to postsynaptic membrane ratio; and (ii) Marked decrease in the toal number of receptors. These

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two factors alone can account for the severely impaired ability of neurally released or microiontophoretically applied ACh to produce depolarization of the endplate. Our data also demonstrate a significant decrease in MEPP frequency in both external and internal intercostal muscles, indicating either that many quanta are released opposite sites where no receptors reside or alternatively that there is an additional presynaptic effect involving a decrease in the number of releasable quanta of ACh. However, the large decrease in MEPP amplitude, combined with the loss of very small MEPPs within the noise of the recording system (I 35 pv), suggests that the decrease in MEPP frequency observed in our preparations is due primarily to postsynaptic effects. Furthermore, the apparent inability of the transmitter to reach the postjunctional membrane in sufficient quantity to activate the appropriate number of receptors makes it very difficult to draw definite conclusions regarding either quanta1 size or amplitude of the potentials. Although morphologic alterations were observed in the endplate region of myasthenic patients, measurements of membrane potential, extrajunctional ACh sensitivity, and lack of tetrodotoxin-resistant action potentials disclosed no major differences from normal muscles. Thus, myasthenic muscle does not show evidence for denervation phenomena. The absence of extrajunctional ACh sensitivity in itself provides further evidence that the alterations of the postjunctional membrane are rather specific in that they involve primarily the ACh receptors. The electron microscopic examination of the endplate regions of myasthenic patients confirms previous studies demonstrating gross alteration of junctional folds and the appearance of globular and membranous material in the greatly widened synaptic cleft (16, 28). Furthermore, we have presented evidence for the possible occurrence of a generalized immune response at the motor endplate. Several granular and agranular leucocytes were observed within 30 to 100 pm of the endplate, both in thin sections and in freeze-fracture replicas. Cells of these several types have been implicated in the production of IgG and IgM antibodies and as phagocytic macrophages [see chapters 3 and 5 in (31)]. The progressive destruction of junctional folds is correlated with the proportional appearance of globular and membranous remnants which appear to be maintained in situ for some time by the regular infoldings of the basal lamina. A strong similarity was noted between negatively and positively stained images of IgG- and IgM-labeled viruses (21, 23, 24) and the very thin smooth and fuzzy coats observed on degenerating junctional fold remnants. Whether these, in fact, represent evidence for an autoimmune response (25) or only nonspecific states of junctional fold degeneration cannot be firmly established. However, observations of cells in vitro undergoing senescence and

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autolysis do not yield comparable images for membrane or cytoplasmic degradation (Rash and Gey, unpublished observations). Therefore, we tentatively suggest that these images provide limited evidence supporting an immunological mechanism of junctional fold destruction, a mechanism compatible with an autoimmune response. The possibility that these alterations of the postjunctional membrane are caused by prolonged treatment with anticholinesterase drugs (15) has been considered. However, patients not receiving anticholinesterase therapy demonstrated identical alterations of the postjunctional folds. Therefore, the profound degenerative changes do not reflect an anticholinesterase effect, at least not from therapeutically administered anticholinesterase. In conclusion, the present study on internal and external intercostal muscles of myasthenic patients show that : (i) Nerve stimulation of a group of about 25% of the surface fibers have EPPs of sufficiently large amplitude to trigger an action potential; another group of fibers had EPPs only, and a third group had no detectable EPPs or spontaneous transmitter release. (ii) The amplitude and frequency of the spontaneous MEPPs were markedly decreased in many surface fibers, particularly in those fibers in which no transmission was observed. (iii) No extrajunctional sensitivity to AC11 was detected in myasthenic muscles. (iv) The observed electrophysiological changes can be correlated with alterations in postsynaptic (and presynaptic) components. It is therefore suggested that in myasthenic muscles the alterations in geometry of the fold are accompanied by a decrease in ACh receptor density, thus accounting for the observed electrophysiological alterations. REFERENCES 1. ALBUQUERQUE, E. X., E. A. BARNARD, C. W. PORTER, and J. E. WARNICK. 1974. The density of acetylcholine receptors and their sensitivity in the postsynaptic membrane of muscle endplates. Proc. Nat. Acad. Sci. lJSA 71 : 2818-2822. 2. ALBUQUERQUE, E. X., J. F. LEBEDA, S. H. APPEL, R. AL&ION, F. C. KAUFFMAN, R. F. MAYER, T. NARAHASHI, and J. Z. YEH. 1975. Effects of normal and myasthenic serum factors on innervated and chronicahy denervated mammalian muscles. Ann. N.Y. Acad. Sci. (in press). 3. ALBUQUERQUE, E. X., and R. J. MCISAAC. 1970. Fast and slow mammalian muscles after denervation. &rp. Ncz~vol. 26: 183-202. 4. ALBUQUERQUE, E. X., and J. E. WARNICK. 1972. The pharmacology of batrachotoxin. IV. Interaction with tetrodotoxin on innervated and chronically denervated rat skeletal muscle. J. Pl~arnzocal. E.rp. Thrv. 180: 653-697. 5. ALMON, R. R., C. G. ANDREW, and S. H. APPEL. 1974. Serum globulin in myasthenia gravis : inhibition of a-bungarotoxin binding to acetylcholine receptors. Srirnce 186 : 55-57. 6. BARNARD, E. A., J. 0. DOLLY, C. W. PORTER, and E. X. ALBUQUERQUE, 1975. The acetylcholine receptor and the ionic conductance modulation system of s1ce1eta1 muscle. Exp. N~urol. 48 : l-28.

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7. BIRKS, R., H. E. HUXLEY, and B. KATZ. 1960. The fine structure of the neuromuscular junction of the frog. J. Physiol. (London) 150: 134-144. 8. COUTEAUX, R., and M. PEC~T-DECHAVASSINE. 1968. Particularit& structurales du sarcoplasme sousneural. C. R. Acad. Sci. Ser. D 266: 8-10. 9. COUTEAUX, R., and M. PECOT-DECHAVASSINE. 1970. Vesicules synaptiques et poches au niveau ‘zones actives’ de la jonction neuromusculaire. C. R. Acad. Sci. Ser. D 271: 2346-2349. 10. DAHLBACK, O., D. ELMQVIST, T. R. JOHNS, S. RADNER, and S. THESLEFF. 1961. An electrophysiologic study of the neuromuscular junction in myasthenia gravis. J. Physiol. (Lowdon) 1586 : 336-343. 11. DREYER, F., and K. PEPER. 1974. Iontophoretic application of acetylcholine: Advantages of high resistance micropipettes in connection with an electronic current pump. PfEugers Arch. 348: 263-272. 12. ELLISMAN, M. H., J. E. RASH, L. A. STAEHELIN, and K. R. PORTER. 1976. Studies ‘of excitable membranes II. A comparison of specializations at neuromuscular junctions and nonjunctional sarcolcmmas of mammalian fast and slow twitch muscle fibers. J. Cell Biol. 68: 752-774. 13. ELMQVIST, D., W. W. HOFMANN, J. KUGERBERG, and D. M. J. QUASTEL. 1964. An electrophysiological investigation of neuromuscular transmission in myasthenia gravis. J. Physiol. (London) 174: 417-434. 14. ELMQVIST, D., and D. M. J. QUASTEL. 1965. A quantitative study of end-plate potentials in isolated human muscle. J. Physiol. (London) 178: 50.5-529. 15. ENGEL, A. G., E. H. LAMBERT, and T. SANTA. 1973. Study of long-term anticholinesterase therapy. Effects on neuromuscular transmission-and on end-plate fine structure. Nczlrology 23 : 1273-1281. 16. ENGEL, A. G., and T. SANTA. 1973. Motor endplate fine structure. Quantitative analysis in disorders of neuromuscular transmission and prostigmine-induced alterations, pp. 196-228. In “New Developments in Electromyography and Clinical Neurophysiology,” Vol. 1, M. E. Desmedt [Ed.]. Karger, Basel. 17. FAMBROUGH, D. M., D. B. DRACHMAN, and S. SATYAMURTI. 1973. Neuromuscular junction in myasthenia gravis : Decreased acetylcholine receptors. Science 182 : 293-295. 18. FAMBROUGH, D. M., and J. E. RASH. 1971. Development of acetylcholine sensitivity during myogenesis. Devel. Biol. 36: 5.5-68. 19. FERTUCK, H. C., and M. M. SALPETER. 1974. Localization of acetylcholine receptor ar-bungarotoxin binding at mouse motor endplate. Proc. Nat. by ‘“I-labeled Acad. Sci. USA 71: 1376-1378. 20. FERTUCK, H. C., and M. M. SALPETER. 1976. Quantitation of junctional and extrajunctional acetylcholine ‘receptors by electron microscope autoradiography after 1261-oi-bungarotoxin binding at mouse neuromuscular junctions. J. Cell. Biol. 69 : 144-158. 21. HAMPAR, B., K. C. HSU, L. M. MARTOS, and Y. L. WALKER. 1971. Serologic evidence that a herpes-type virus is the etilologic agent of heterophile-positive infectious mononucleosis. Proc. Nat. Acad. Sci. USA 68: 1407-1411. 22. HEUSER, J. E., and T. S. REESE. 1973. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell. Biol. 57 : 315344. 23. HOGLUND, S. 1967. Electron microscopic investigations of the interaction between the TZ-phage and its IgG- and LgM-antibodies. Virology 32: 662-677. 24. LAFFERTY, K. J., and S. OERTELIS. 1963. The interaction between virus antibody III. Examination of virus-antibody complexes with the electron microscope. Virology 21: 91-99.

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1973. Autoimmune response to acetylcholine J., and J. LINDSTR~M. receptor. Scic+bcc 180 : 871-872. RASH, J. E., and M. H. ELLISMAN. 1974. Studies of excitable membranes. I. Macromolecular specializations of the neuromuscular junction and the nonjunctional sarcolemma. J. Cc/l Biol. 63: 457-586. RASH, J. E., J. E. WARNICK, M. H. ELLISMAN, and E. X. ALBUQUERQUE. 1975. Freeze-fracture stereoscopy of quiescent, stimulated and toxin-treated neuromuscular junctions. /. Cell Biol. 67: 354a. SANTA, T., A. G. ENGEL, and E. H. LAMBERT. 1972. Histome,tric study of neuromuscular junction ultrastructure. Nctlrology 22 : 71-82. STRAUSS, A. J. L., B. C. SEAGAL, K. C. Hsu, P. M. BURKHOLDER, W. L. NASTUK, and K. E. OSSERMAN. 1960. Immunofluorescence demonstration of a muscle binding, complement-fixing serum globulin fraction in myasthenia gravis. Proc. Sot. Ex/J. Biol. Med. 105: 184-191. STRAUSS, A. J. 1968. Myasthenia gravis autoimmunity and th,e thymus. A&. Inter. Med. 14: 241-280. TANAKA, J., and J. R. GOODMAN. 1972. “Electron Microscopy of Human Blood Cells.” Harper and Row, New York. PATRICK,