Electrophysiological studies of thymectomized and nonthymectomized acetylcholine receptor-immunized animal models of myasthenia gravis

EXPERIMENTAL

NEUROLOGY

63, l-27 (1979)

Electrophysiological Studies of Thymectomized Nonthymectomized Acetylcholine ReceptorImmunized Animal Models of Myasthenia Gravis W.D.

Departments

and

NIEMI, W. L. NASTUK, H. W. CHANG, A. S. PENN, AND T. L. ROSENBERRY' of Physiology

and Neurology, Received

Columbia May

University,

New

York

10032

2. 1978

White New Zealand rabbits were immunized with acetylcholine receptor (AChR) purified from the electric organs of either the Electrophorous electricus (eel) or Torpedo californica (torpedo). Approximately 4 days after a second injection of 100 pg AChR in complete Freund’s adjuvant, the immunized animals developed weakness of the peripheral muscles which was aggravated by repetitive activity and was partially alleviated by anticholinesterases such as edrophonium or pyridostigmine. Electromyograms taken from the anterior tibialis muscle demonstrated a decrement of the compound action potentials at a stimulation frequency of 5 Hz which was reversed by the intravenous administration of 1 mg pyridostigmine. If the animals were not treated with antichohnesterases they died within a couple of days. Biopsies of the intercostal muscle were done under Nembutal anesthesia, and intracellular recordings revealed subliminal end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) of greatly diminished amplitudes. At some neuromuscular junctions no MEPPs were detected, and in other junctions MEPPs were seen but no EPP was evoked upon nerve stimulation. Muscles from two immunized rabbits showed spontaneous muscle action potentials occurring at 1 to 2 Hz. Serum from the rabbits immunized with torpedo or eel AChR, when applied to in vitro preparations of the eel electroplaque or frog Abbreviations: AChR-acetylcholine receptor; EMG-electromyogram; MEPPminiature end-plate potential; MG-myasthenia gravis; CAP-compound action potential; carb-carbamylcholine, PJM-postjunctional membrane. 1 This work was supported by grants from the National Institutes of Health (NS04988) and Muscular Dystrophy Association to W.L.N. and a Kermit Osserman Fellowship to W.D.N. from the Myasthenia Gravis Foundation. The authors thank Dr. R. E. Lovelace for helping with the EMGs, Dr. George Toufexis and Mr. Kelvin Brockbank for helping with the thymectomies, and Dr. A. Hays for the histological examinations of the thymus tissue.

0014-4886/79/010001-27$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

NIEMI ET AL. cutaneous pectoris muscle, caused a decrease in postjunctional membrane sensitivity to carbamylcholine and reduced the MEPP and EPP amplitudes even after heat inactivation of the serum complement (56°C for 30 min). In addition to a decrease in chemosensitivity, an enhancement of receptor desensitization occurred in the neuromuscular junctions of serum-treated frog muscles. Thymectomies on adult rabbits prior to the immunization had the effect of delaying the onset of myasthenia in two of six rabbits, and in three of six rabbits only mild myasthenia occurred. The blood antibody titers to AChR were similar in both the thymectomized and nonthymectomized rabbits.

INTRODUCTION The hypothesis that myasthenia gravis (MG) is an autoimmune disease (14,24) has recently received further support from the observation that the immunization of animals with pure acetylcholine receptor protein (AChR) produces a condition in the animals which resembles human myasthenia gravis. The use of purified acetylcholine protein (17,25) as an antigen was a significant improvement over the earlier immunization attempts with crude cardiac muscle extract (20) or thymus extracts (6) in generating experimentally reproducible symptoms of myasthenia. The recent literature has provided clinical and electrophysiological evidence in support of the AChR-immunized animal as a model for MG. How closely this experimental model resembles human MG was recently the subject of intensive investigations in a number of laboratories (4, 8, 19, 22). The AChR-immuaized animal shows severe muscular weakness which appears first in the hind legs and then spreads rostrally. This weakness is aggravated with repetitive activity and occurs a few days following the second injection of 100 pg of either eel or torpedo cholinergic receptor protein in complete Freund’s adjuvant. Compared to human MG, the experimentally induced myasthenia develops more rapidly and has a higher fatality rate (>90%). The main locus of the pathological derangement in these animals appears to be at the neuromuscularjunction (4,8, 19,22). In AChR-immunized rats morphological disruption of the neuromuscular junction was shown to occur concurrently with the presence of lymphocytes and macrophages at the junctional region (4). In human MG the binding of the C3 serum complement component to the postsynaptic membrane was demonstrated (2). The passive transfer of myasthenia to normal mice was demonstrated with serum from MG patients (27, 28). In an analogous manner, the application of serum from immunized rabbits to normal frog muscle (7) or to normal eel electroplaque (18, 25) severely depressed postjunctional membrane sensitivity to cholinergic agonists. In addition, according to TarrabHazdai er al. (26) and Lennon ef al. (12) lymph node cells from AChRimmunized animals also transferred the disease to normal animals. Indirect evidence that the physiological lesions in MG may also involve cell-

ANIMAL

MODELS

OF MYASTHENIA

GRAVIS

3

mediated immunity was indicated by the presence of lymphocytes sensitized to AChR in patients with myasthenia (21) as well as in receptorimmunized guinea pigs (26). In the present study we determined the contribution humoral immune factors make to the pathogenesis of MG. In particular we wished to determine if the mere binding of cholinergic receptor antibodies to the postjunctional receptor sites could produce the physiological changes characteristic of MG. The ability of the externally applied antibody to disrupt neuromuscular transmission in normal muscle, the effect of antireceptor antiserum on cholinergic receptor desensitization, and recovery from desensitization were also investigated. We also evaluated the effect of thymectomies on adult rabbits prior to the immunization and myasthenia, because thymectomy is a common therapeutic mode in MG. MATERIALS

AND METHODS

Immunization. White New Zealand rabbits, 2.7 to 3.2 kg, (6 to 7 lbs.) were inoculated subcutaneously in the back at four sites with a total of 100 pug (0.25 ml) acetylcholine receptor protein isolated from Electrophorous electricus and Torpedo californica (1) emulsified in 0.25 ml complete Freund’s adjuvant. Fifteen rabbits received eel AChR and eight received torpedo AChR. One month later these rabbits were inoculated again, using the same regimen. The animals were examined daily, and blood samples were drawn from ear veins at various intervals. Antibody Assays. Antibody titers were determined using radioimmunoassay (18). AChR (0.5 mg) was allowed to react with 1251-labeled-a-bungarotoxin (12 nmol toxin/mg receptor) for 2 h at room temperature. Toxin-receptor complex containing 6 pmol of toxin binding sites was then incubated with serial dilutions of rabbit antiserum in 0.1% Triton, 0.1 M NaCl, 0.02 M phosphate buffer, pH 7.2 (total volume, 200 ~1; 0.1% Triton-phosphate-buffered saline), overnight at 4°C. Immune precipitates were recovered after centrifugation at 3000 g, washed twice with the above buffer, then dissolved in 0.4 ml 0.1 N NaOH. The solubilized precipitate and portions of supernatant and wash were counted in Scintisol. Control samples were prepared with normal rabbit serum to assess any binding by the normal serum and to correct for nonspecific absorption of the label to glass or precipitate. The titer of antibody to AChR was measured as the number of nanomoles toxin-AChR precipitated per microliter antiserum added, and expressed as nanomoles of toxin binding sites precipitated per milliliter antiserum. Thymectomies. Six normal rabbits which had been under observation

4

NIEMI

ET AL.

for a week and shown to be in good health were thymectomized under Nembutal anesthesia using an open sternum approach. The thymus was removed by blunt dissection using a small gauze ball held by forceps, thereby avoiding damage to the pleural or pericardial membranes. The rabbits then recovered for 3 weeks before immunizations were begun. Tissue from the thymus region was taken postmortem and examined histologically to confirm the absence of the thymus. Electromyogrums. Weekly electromyographic recordings (EMG) were made on the immunized rabbits. EMGs were taken using a silver plate electrode placed firmly against the well-shaved skin over the anterior tibialis muscle using electrocardiogram paste to improve electrical contact. The sciatic nerve was stimulated with two Teflon-coated needle electrodes inserted through the skin 1 cm apart into the sciatic notch. Five rectangular pulses of 0. I-ms duration (at two times threshold) were delivered at frequenceis of 2,5, 10,20, and 50 Hz. The EMG decrement was expressed in terms of the percentage decrease in the amplitude of the third or fifth compound action potential (CAP) measured with respect to the amplitude of the first CAP of that particular train. The SO-Hz train was allowed to continue 10 s to study posttetanic potentiation and exhaustion. At the end of the lo-s, 50-Hz train, the stimulus frequency was reduced to 0.1 Hz and was maintained at this value until the CAP amplitude stabilized at a constant value. Posttetanic potentiation was determined from the maximum percentage increase in CAP amplitude which occurred during this lowfrequency train measured with respect to the first CAP of the 50-Hz train. Posttetanic exhaustion was calculated from the minimum CAP amplitude observed during the O.l-Hz train. Thirty minutes after these initial recordings were obtained, edrophonium (Tensilon) was given via an ear vein, and a similar series of EMG measurements were made to test the animal’s responsiveness to the anticholinesterase. Efectrophysiology . Electrophysiological studies were conducted using the frog cutaneous pectoris muscle pinned out on Sylgard. The dorsal surface of the muscle was placed face up, and the nerve was arranged for stimulation by passing it through two stainless-steel coils embedded in a small polyethelene tube. The rabbit serum used in most electrophysiological experiments involving frog muscle was heat-inactivated (unless otherwise stated) 30 min at 56°C followed by dialysis against a frog Ringer’s solution consisting of 112.4 mM Na, 2.5 mM K, 1.8 mM Ca, 117.1 mrvr Cl, and 3 mM HEPES (IV-2-hydroxyethylpiperazine-IV’-2-ethanesulfonic acid) and adjusted to pH 7.4 with NaOH. Such serum was applied undiluted to single frog end-plates using lOO-pm micropipets at a flow rate of 1 to 2 fil/min. The in vitro electrophysiological studies on the rabbit intercostal muscle

ANIMAL

MODELS

OF MYASTHENIA

GRAVIS

5

were carried out after the EMG recordings were completed. The excised muscles were placed into cold oxygenated modified Liley’s solution (HEPES buffer was substituted mole for mole for bicarbonate in Liley’s solution which was oxygenated with 100% 0,). The muscle bundle was dissected to provide a thin layer of fibers containing a nerve twig. The experiments were then carried out at room temperature (25°C) with the muscle sheet pinned out on clear Sylgard and transilluminated. Action potentials were generated at nonjunctional membrane areas by direct stimulation via a second intracellular glass microelectrode inserted approximately 1 mm from the recording electrode. The eel electroplaques were dissected and mounted according to the method of Schoffeniels (23) and bathed in an eel Ringer’s solution (30). The intracellular potentials were measured with lo- to 20-MQ glass microelectrodes, and the electroplaques were stimulated via external Ag-AgCl electrodes. Rabbit serum was diluted in eel Ringer’s and placed 0.5 h in the pool which faced the innervated side of the cell. The rabbit serum used on electroplaques was neither heat-inactivated nor dialyzed against eel Ringer’s, only diluted. Electroplaque cholinergic chemosensitivity was tested intermittently by replacing the bathing solution with one containing the appropriate concentration of carbamylcholine (carb) and measuring the maximum depolarization that occurred in 2 min. Sensitivity Measurements. Postjunctional membrane sensitivity measurements on the frog muscle were made either iontophoretically, using fine glass micropipets (having a resistance of 100 to 200 Ma and filled with 3 M carb) or with perfusions of solutions containing 20 PM carb. Iontophoretic currents of 10 to 50 nA with durations of 3 to 700 ms were used for the iontophoresis and were monitored on a second channel across a 50-K resistor in series with ground. Desensitization. Desensitization was measured using iontophoretic current pulses applied at 1 Hz to produce depolarizations 500 to 700 ms in duration and several millivolts in amplitude. The magnitudes and the durations of the iontophoretic pulses used in the desensitization experiments were adjusted for each cell individually. Measurements were made before and after the cells were perfused with antiserum. The maximum and minimum depolarizations that occurred at the beginning and the end of the test train were used to calculate the percentage decrease in depolarization produced by carb (see Fig. 6). Desensitization was measured during the first 30 s of the I-Hz train of pulses, and recovery from desensitization was monitored for a few minutes after the cessation of the l-Hz train. The time (T& needed to reach a level of depolarization equal to one-half the maximum depolarization produced, and the time to reach a plateau of steadystate depolarization (T,,) were determined. In some determinations dif-

6

NIEMI

ET AL.

ferent muscle fibers were used for the control and test determinations to ascertain that no residual effects were produced during the control determinations, and in many the same fiber was used for both control and test measurements. This is referred to in the tables as nonpaired and paired, respectively, for the two methods. RESULTS Evidence of Muscular Weakness in Receptor-immunized Rabbits. Three days after the immunized rabbits had received a second injection of 100 pg AChR in complete Freund’s adjuvant, they became lethargic, and developed a severe muscular weakness on the fourth to sixth day. The weakness seemed to predominate in the limbs, neck, and thoracic muscles. The skeletal muscle paralysis progressed rostrally, and by day 7 the animals died, most likely from respiratory paralysis. Autopsies on the rabbits were unremarkable, though two of ten animals autopsied had fluid accumulation in the lungs. Little difference in the outcome of the immunization was noted between those rabbits that were injected with torpedo receptor and those with Electrophorous receptor protein except that the “torpedo rabbits” developed myasthenia 1 to 2 days later than did the “Electrophorous rabbits.” In all cases, the two-injection regimen of immunization eventually proved fatal, except for two rabbits that were manually fed and treated with pyridostigmine for 1 week during the period of severe myasthenia. These rabbits (IX and XI) then lived 8 months with no reappearance of myasthenia and died from a Pasteurella infection. EMGs on immunized rabbits having evidence of muscle weakness showed large compound action potential (CAP) decrements at stimulation frequencies of 5 and 10 Hz (Table 1). These decrements showed improvement after the intravenous administration of edrophonium (1 mg/2.7kg rabbit); the CAP decrement was maximally reversed 3 min after the anticholinesterase injection. The CAP decrement in anterior tibialis of an immunized rabbit at stimulation of 5 and 10 Hz before and after edrophonium administration was 37.5 and 7%, respectively (Fig. 1). Control rabbits (nonimmunized) showed no significant compound action potential decrement at stimulation frequences of 5, 10, or 20 Hz. Posttetanic potentiation, when seen, was observed only in the AChRimmunized animals. Posttetanic potentiation was seen in 4 of 12 immunized rabbits after 50-Hz stimulation, and it ranged from 7 to 64%. Anti-AChR Antibody Titers. Antibody titers against purified eel and torpedo AChR were determined using 1251-labeled toxin-labeled receptor and radioimmunoassay. All immunized rabbits were found to have signifi-

ANIMAL MODELS OF MYASTHENIA GRAVIS

7

TABLE 1 Electromyograms of Thymectomized and Nonthymectomized Rabbits Immunized with Acetylcholine Receptor Protein from Eel or Torpedo” Decrement Before

Group

Frequency (Hz)

of compound

action

potential

edrophonium Third potential

(%)

AtIer Fifth potential

Frequency (Hz)

edrophonium Third potential

Fifth potential

No. of animals

Nonthymectomized and immunized with eel AChR

2 5 10 50

23.0 47.0 56.0 65.0

+ 5.86 2 11.2 + 8.9 -c 9.3

41.0 64.0 75.0 82.6

t k + +

4.3 9.2 6.5 7.2

2 5 10

11.6 5 22.0 + 27.0 c

2.1 3.7 3.4

27.4 + 35.0 2 43.0 r

6.5 6.4 6.6

5 5 5

Nonthymectomized and immunized with torpedo AChR

2 5 10 50

27.2 45.8 47.5 60.0

f 5.0 r 8.2 + 11.1 k 10.3

44.0 68.5 74.8 81.2

+ f t t

9.5 9.4 7.8 8.9

2 5 10

5.6 c 7.6 + 11.5 -’

2.1 3.8 6.4

8.7 + 9.3 f 18.8 2

3.2 5.4 5.6

3 3 3

Thymectomized and immunized with torpedo AChR

2 5 10 20 50

29.3 37.7 41.3 48.0 48.3

k + f + c

10.7 16.2 19.6 20.9 20.8

45.0 57.7 58.3 61.3 68.7

+ c ” -c c

13.2 20.3 11.4 25.3 22.4

2 5 10 20 50

5.1 6.0 15.4 24.2 29.6

Nonthymectomized and nonimmunized

2 5 10 20 50

0.3 0.8 0.4 2.0 6.0

2 2 -’ k C

0.2 0.7 0.5 0.7 1.7

0.8 1.0 1.8 4.0 12.5

? * * t +

0.7 0.9 1.2 0.9 2.5

z 2.1 f 1.2 f 7.6 2 10.1 T 12.4

10.9 13.6 21.3 30.0 36.3

+ 7.7 + 12.9 f 9.8 f Il.9 f 14.2

3c 3 3 3 3 4 4 4 4 4

(’ Electromyograms were obtained from the anterior tibirdis muscle of rabbits (immunized with AChR, or thymectomized and immunized). using a silver plate electrode over the shaved skin and a needle electrode inserted into the distal tendon. The percentage decrement of the third and fifth compound action potential is given relative to the first potential. No significant difference West. P < 0.05) was found between the thymectomized and nonthymectomized rabbits immunized with torpedo AChR. L Mean + SE. c In the thymectomized group. two eel-immunized rabbits and one torpedo-immunized rabbit did not develop myasthenia after the second injection of AChR, but died suddenly 1 week later from unknown causes. All nonthymectomized rabbits developed myasthenia within 5 days after the second injection.

cant antibody titers to AChR using the same antigen for the immunizations and the assays (Table 2). The animals were divided into two groups, thymectomized and nonthymectomized. The anti-AChR titers in the thymectomized group ranged from 0.28 to 1.95 nmol toxin-binding sites precipitated by 1 ml antiserum. The titers in the nonthymectomized group varied from 0.75 to 4.5. The nonimmunized controls had background titers from 0.01 to 0.05 nmol/ml, which is ten times lower than the lowest titer seen in the immunized

animals.

Intracellular Recordings. Intracellular recordings were done on intercostal biopsies from both control and AChR-immunized rabbits. The most striking alteration noted in the muscles of immunized animals was a large decrease in the amplitude of the miniature end-plate potentials (MEPPs)

NIEMI

a

ET AL.

ANIMAL

MODELS

OF MYASTHENIA TABLE

2

Serum Antibody Titers and Condition of AChR-Immunized either Thymectomized or Nonthymectomizeda

Rabbit

Antigen

Nonthymectomized D J N 1, VII IVb Controls

Torpedo Eel Eel Eel -

Thymectomized C E F H K L

Eel Eel Torpedo Torpedo Torpedo Torpedo

Eel

Antibody titerb (nmohml) 1.03 4.50 2.25 1.2

1.1 0.75

EMG Decrement” (% at 5 Hz)

++++ ++++ +++

50

++++ +++ ++++

No MG symptoms

1.05 0.88

No MG symptoms No MG symptoms +++ No MG symptoms ++

0.28 2.00

Rabbits

Myasthenia

0.01 * 0.05

1.01 1.95

9

GRAVIS

++++

55 60 0+5

60 30 75

n Rabbits that were immunized with AChR purified from electric eel or Torpedo californica were tested for muscle strength by tugging at their legs and/or gauging their postures. Rabbits that laid on their sides and did not have the ability to right themselves were considered + + + + . All rabbits showing weakness responded to edrophonium (Tensilon). b Expressed as the number of toxin-binding sites precipitated by 1 ml rabbit serum. e The amplitude of the fifth potential + amplitude of the first x 100.

(Fig. 2). In some of the immunized rabbits, MEPPs were absent in all fibers sampled. The mean MEPP amplitude (*SD) of those recorded from immunized animals was 0.27 + 0.17 vs. 0.54 k 0.21 mV for the control animals (Table 3). The former figure is an underestimation as many neuromuscular junctions from immunized rabbits (verified by cholinesterase staining) showed no detectable MEPPs, and these could not be included in the averages. Despite this, the measured decrease in MEPP amplitude is highly significant. The histogram in Fig. 3 shows an example of the differFIG. 1. Electromyograms (EMG) from the anterior tibialis muscle of a rabbit which had been immunized twice with 100 pg acetylcholine receptor protein from torpedo. A-compound action potentials at a stimulation of 5 Hz from rabbit T7 4 days after receiving a second injection of 100 pg AChR and showing a decrement of 37.5%. B-same rabbit 5 min after receiving 1 mg edrophonium via ear vein and showing a decrement of only 7%. C-same as A but taken at 10 Hz. D-same as B but taken at 10 Hz. Calibration: 1 large division = 1 s or 10 mV.

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ET AL.

FIG. 2. Intracellular recordings of miniature end-plate potentials (MEPPs) from intercostal muscle of normal rabbits and rabbits which have been immunized with acetylcholine receptor protein purified from either electric eel or Torpedo cali’ornica. A-MEPPs from a normal rabbit; B-MEPPs from a rabbit immunized with “torpedo protein”; C-MEPPs from a rabbit immunized with “eel protein.” Calibration: 0.625 mV, 5 s/division.

ence seen in MEPP amplitude distribution recorded from muscle fibers of two different rabbits, (a) from an immunized rabbit and (b) from a control rabbit. In the immunized animal the MEPP amplitude distribution was shifted

ANIMAL

MODELS

OF MYASTHENIA TABLE

Miniature

3

End-Plate Potentials (MEPPS) in Acetylcholine

Group

MEPPs (mV)

Immunized Nonimmunized

Receptor-Immunized

Frequency (mini)

0.27 f 0.17O 0.54 2 0.21

11

GRAVIS

Rabbits”

No. of rabbits/fibers

70.1 2 72 24.8 f 1.5

4/10 3110

a Only the MEPP amplitudes of the two groups are significantly different, (P < 0.01) using a t-test. Four other immunized rabbits had no detectable (co.1 mV) MEPPs in the muscle biopsy sampled and were not included in the above results. * Mean ? SD.

to the left. Using the modal value and comparing the MEPP distributions of the immunized and nonimmunized animals, one sees a fourfold diminution in amplitude. Comparing the means of the 10 largest MEPPs from each group we obtained 0.47 + 0.13 and 1.37 k 0.38 mV for the immunized animal and the control, respectively, which represents a threefold reduction. Thus, a severe reduction in MEPP amplitude occurred in the immunized animals, although we found some MEPPs from myasthenic muscles that were in the normal range, i.e., ~1 mV. Effect of Anti-AChR Antisera on Tissue Preparations in Vitro. All fif0

A

0

0.5 MEPP

AMPLITUDE

1.0 (mv.1

15 MEPP

AMPLITUDE

(mv)

FIG. 3. Histogram of miniature end-plate potential (MEPP) amplitudes recorded from the intercostal muscle of a control rabbit (A) and of an AChR-immunized rabbit (B). The mean MEPP amplitude of the immunized rabbit was 0.25 mV 5 days after the second injection of receptor whereas in the control rabbit it was 0.67 mV. The histogram becomes more skewed andis shifted to the left in B. The data are expressed in terms of the percentage of total MEPPs which occurred in each millivolt bin. The left histogram was based on 208 MEPPs, and the right histogram was based on 205 MEPPs.

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teen eel AChR-immunized and eight torpedo AChR-immunized rabbits developed severe myasthenia and were bled at various intervals. Sera obtained from rabbits with severe myasthenia were applied to in vitro preparations of eel electroplaques and frog cutaneous pectoris. Seven antieel antisera and five anti-torpedo antisera were either bath-applied or perfused onto the above preparations. The effect of the applied antisera on the action potential amplitude of either frog muscle or electroplaque was not significant, but a reduction in the maximum rate of rise of the electroplaque action potential occurred, whereas no change occurred in that of the frog. The antisera applied to frog muscle was heat treated (56°C 30 min) and that applied to the electroplaque was not (Table 4). The most dramatic effects of antiserum treatment were at the synapses. The serum reduced the end-plate potentials to the point where they could no longer trigger action potentials. The postsynaptic membrane was tested for its chemosensitivity which was greatly reduced. Chemosensitivity determinations with the eel electroplaques are summarized in Table 5. The anti-eel antisera reduced the sensitivity of the innervated membrane to bath-applied carb by 55%, and the anti-torpedo antisera inhibited the electroplaques by 41%. The amount of inhibition ranged from 32 to 89% depending on which serum was being tested. The inhibition was only slightly reversed by washing the preparation in normal Ringer’s solution. A typical experiment is demonstrated in Fig. 4A. The maximum depolarization produced by a solution of 50 PM bath-applied carb is plotted TABLE

4

Effect of Antireceptor Antibodies on Extrajunctional Membrane Properties of the Rabbit Muscle, Frog Muscle, and Eel Electroplax” Resting potential (mv)

Action potential (mV)

Overshoot WJ)

Maximum rate of rise (V/S)

Maximum rate of fall WS)

iv”

CO”trOl Rabbit intercostal Eel electroplax Frog cutaneous pectoris

70.0 e 8.5’ 84.6 -c 5.4 89.9 + 2.0

105.9 + 6.2 136.7 k 10.0 127.0 c 4.0

35.9 T 3.6 52.1 c 3.2 41.0 t 10.0

357.2 ? 76 360.0 T 115 410.0 + 73

91.4 -+ 13.3 141.7 2 33.0 117.0 -c 22.0

1012 4 7lw7

Test Rabbit intercostal Eel electroplax Frog cutaneous pectoris

71.2 + 12.5 90.7 k 6.6 87.6 k 6.0

88.5 e 15.0 119.9 + 14.0 128.0 k 8.0

17.2 f 29.2 + 40.2 *

231.2 + 126 219.0 k 118 418.0 2 100

85.3 f 36.0 99.0 + 20.0 121.9 f 12.0

IO/2 4 7w7

8.9 5.3 7.0

L1The rabbit intercostal muscle was biopsied from immunized rabbits under Nembutal anesthesia and was not subsequently exposed to any antiserum in virro. Normal cells from eel electric organ and from frog cutaneous pectoris muscle were incubated I h in a Ringer’s solution containing a 1: 10 dilution of the antiserum from immunized rabbits. Rabbit antiserum applied to the frog muscle only was heat treated 30 min at 56°C. B Number of cells/number of muscles. c Mean -r SD.

ANIMAL

MODELS

OF MYASTHENIA TABLE

13

GRAVIS

5

Effect of Rabbit Anticholinergic Receptor Antiserum on the Average Sensitivity of the Electroplax to Carbamylcholine” Millivolt Serum type Anti-eel Anti-torpedo Normal

depolarization

Control

Test

No. of cells

No. of sera

t

31.5 k 6.P 30.6 k 4.4 33.5 k 6.6

13.9 + 5.6 18.1 + 7.2 27.8 + 4.8

10 9 4

7 5 2

9.5, P < 0.01 4.5,P < 0.01 1.4, P > 0.1

a The serum from either eel receptor or torpedo receptor-immunized rabbits, or from nonimmunized rabbits (normal) was diluted 1: 10 in eel-Ringer’s solution and bath-applied to the innervated face of the electroplax for 0.5 h, and the millivolt depolarization produced by a 30 PM carbamylcholine solution was compared to the depolarization produced by the same concentration of carbamylcholine just prior to the serum application. The number of cells used and the number of different rabbit sera from different immunizations are indicated. A paired t-test indicated that all test groups were significantly different from controls. A significant reduction in sensitivity was produced by the normal serum (paired t test). * Mean + SD.

at various times after exposure to anti-AChR antiserum. These points were taken at 33, 54, 73, and 89 min after the application of diluted antiserum (1:20) to the innervated face of the eel electroplaque. Under these conditions, the reduction in chemosensitivity developed relatively slowly; approximately 0.5 h of exposure to serum diluted to 1:20 is required to reduce the carb depolarization by 50%. The maximum depolarization of the innervated membrane produced in the electroplaque after exposure to rabbit anti-eel antisera averaged 13.9 I~I 5.6 vs. 31.5 + 6.5 mV (mean t SD) for the serum treated control preparation. Similarly for the rabbit anti-torpedo antisera, the carb depolarization averaged 18.1 + 7.2 mV (mean + SD). Some inhibition was consistently noted with control rabbit sera, and though small, it was found to be statistically significant. Complete inhibition of the response of the electroplaque postjunctional membrane to carb was never achieved with any of the serum samples tested even after prolonged exposure to antiserum (to 3 h). The experiment depicted in Fig. 4B involved three different cells; one cell was exposed to normal rabbit serum diluted 1: 10 in eel Ringer’s, a second cell was exposed to a 1: 10 dilution of rabbit anti-AChR (eel) antiserum, and a third cell was exposed to a 1:5 dilution of the same antiserum. The electroplaque’s chemosensitivity was tested periodically by replacing the antiserum-containing pool with a solution containing carb at different concentrations, beginning 1 h after an initial incubation with the antiserum solution. The concentration-response curves with carb after exposure of the cell to

14

NIEMI ET AL.

I 75

90

MINUTES

CARBAMYLCHOLINE

@MM)

4. A-The maximum depolarizations ofthe postsynaptic membrane produced by bath applications of 50 j&M carbamylcholine (carb) to an eel electroplaque which had been exposed to a 1:20 dilution of immune rabbit serum beginning at time 5 min (dotted line). The cell was periodically tested for its carbamylcholine sensitivity by applying 50 PM carb in eelRinger’s solution for 2-min periods at the times indicated. B-Concentration-response curves ofthree different electroplaques using carb: (0) no antiserum, (0) a 1: 10 dilution ofanti serum, (0) a 15 dilution of antiserum. The antisera were applied for 0.5 h prior to the application of carb. FIG.

ANIMAL

MODELS

OF MYASTHENIA

GRAVIS

15

anti-AChR antiserum shift to the right, and a decrease in the maximum depolarization produced by carb occurs. The latter curves also do not appear to be sigmoidal in shape as is true for the control curve. In the case of the frog postjunctional membrane a faster and more pronounced reduction in sensitivity to carb was obtained because the antiserum was applied undiluted by microperfusion (rather than with bath application of diluted antisera as done in the electroplaque experiments). In a typical result the depolarization response of the postjunctional membrane of frog cutaneous pectoris to 500-ms iontophoretic pulses of carb delivered at 0.016 Hz during the microperfusion with a high-titer rabbit anti-torpedo antiserum, showed a 75% decrease during the first 4 min of perfusion (Fig. 5). Because it was not possible to restore the original response to carb by making small adjustments in the position of the iontophoresis pipet, the decreased response produced by application of antiserum is considered to be real and not a result of a movement artifact. In addition to the above iontophoretic measurements, neuromuscular transmission was examined in anti-AChR antiserum-treated muscles. Neuromuscular junctions were perfused with antisera while the presence or absence of a muscle action potential following nerve stimulation was noted. The results were expressed in terms of the percentage of junctions which did not initiate an action potential (Table 6). In 19 junctions that were perfused with antiserum, subthreshold EPPs were seen in all of them and averaged 16.2 +- 7.4 mV. In eight of 27 junctions the EPP reached threshold even after 0.5 h of antiserum perfusion. Some of these junctions may have been situated facing inward on the underside of the muscle fiber which would make access of externally applied antibody difficult. Perfusions of undiluted rabbit anti-AChR antiserum onto frog end-plates also caused a pronounced reduction in the MEPP amplitude. At end-plates that were perfused with undiluted antiserum, a fourfold decrease in MEPP amplitude was found (Table 7). No change in MEPP frequency was noted. Effect of Thymectomies on the Induction of the Model Disease. Open-sternum thymectomies on six adult rabbits 3 weeks prior to immunization with AChR had the effect of reducing the rate of onset of the myasthenia, but nonetheless, myasthenia still occurred in three of six thymectomized rabbits. These animals showed large CAP decrements and subthreshold end-plate potentials as well as reduced MEPPs. However, three of the immunized rabbits in the thymectomized group did not show any myasthenic symptoms for 1 week after the boost. Two of these rabbits died suddenly during the night when they were not under observation, due to a Pasteurella epidemic in the animal quarters, and one died under anesthesia.

16

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ANIMAL

MODELS

OF MYASTHENIA TABLE

17

GRAVIS

6

Amount of Neuromuscular Block Produced in Frog Neuromuscular Perfused with Undiluted Rabbit Anti-AChR Antiserum”

Muscle

12 3 4 5 6 7 9

Junctions

Junctions with subthreshold EPPs

Antiserum we

Antibody titer (nmol/ml)

(%)

Junctions tested

Anti-eel C Anti-eel I Anti-eel VII Anti-torpedo L Anti-torpedo K Control serum Frog Ringer’s

1.05 1.2 1.1 2.0 0.28 0.10 * 0.05 -

loo 80 33.3 66.6 75.0 0 0

5 5 6 3 8 6 10

D The percentage of junctions that failed to produce an action potential upon nerve stimulation after having been microperfused 0.5 h with undiluted heat-inactivated (WC, 30 min), Ringer’s-dialyzed antiserum is tabulated in the fourth column. Antiserum C was from a rabbit that was thymectomized and immunized with eel-receptor; antiserums I and VII were from an eel-immunized but nonthymectomized rabbit; antiserums K and L were from thymectomized and torpedo-immunized rabbits. The control serums were from nonimmunized, nonthymectomized rabbits. The antibody titers are expressed in terms of nanomoles toxin binding sites precipitated per milliliter rabbit serum.

Electromyograms taken when the immunized animals displayed some muscle impairment revealed CAP decrements characteristic of myasthenia. This was true of both the thymectomized and nonthymectomized groups, all of which had high titers of circulating antibody to acetylcholine receptor. Autopsies and histological examination confirmed the absence of a thymus in the thymectomized animals (Table 1). Postjunctional Membrane Desensitization. It is known that prolonged exposure of the postjunctional membrane to cholinergic agonists, such as carb, results in a loss of chemosensitivity to these agonists, a phenomenon referred to as receptor desensitization (11). We wished to determine FIG. 5. The end-plate membrane depolarization of a frog neuromuscular junction tested by iontophoretic application of carb at intervals during exposure to undiluted rabbit antireceptor antiserum applied locally by superfusion with a glass micropipet at 3 pl/min. The upper trace is the current applied to the iontophoretic pipette. The bottom five traces are depolarizations produced by the same current but at I-min intervals (top to bottom). The antiserum was obtained from a severely paralyzed rabbit that had been immunized with torpedo acetylcholine receptor protein. The antiserum was heat-treated (56”C, 0.5 h) and dialyzed in frog Ringer’s prior to application. Calibration: 2 mV and 100 ms/large division for lower trace, 2.5 nA and 100 msllarge division for upper trace.

18

NIEMI ET AL. TABLE

7

Effect of Rabbit Antireceptor Antiserum on Miniature Potentials (MEPPS) in Frog Muscles” Before Rabbit Serum type

Method of application

Altti-eel (ttontbymectomized) Anti-torpedo (nonthymectomized) Allti-eel (thymectomized) Anti-torpedo (thymectomized)

Bath (I h) (I:20 dilution) Microperfusion (undiluted) Microperfusion (undiluted) Microperfusion (undiluted)

Control Serum

Microperfusion (undiluted)

a A signiticant

diierence

Amplitude (mv)

treatment

End-plate After treatment

Frequency (min.‘)

Amplitude (mV)

0.74 + 0.15c

59 + 33

0.66 + 0.09

0.81 + 0.42

29 + 30

0.1

0.84 + 0.52

13+

1

0.83 f 0.22 0.82 + 0.25 0.84 + 0.52

N

45 + 27 3+

@

2

3

0.17 + 0.02

13 r 14

2

25 + 19 24 + 18

0.24 + 0.11 0.26 + 0.16

21 + 17 23 + 21

11 22

222

0.66 f 0.38

14 + 13

3

7

+ 0.1

Frequency (min-‘1

exists between MEPP amplitudes before and after 0.5-h treatment with undiluted exists between the thymectomized and nonthymectomized groups. b Three to ten muscle fibers from each muscle were sampled, and 2 min of MEPP recording at each junction to calculate the means. c Mean + SD.

antiserum

(P < 0.01). No difference

were used

whether or not the postjunctional membranes of frog muscle fibers treated with rabbit anti-AChR antibodies would show a difference in their susceptibility to desensitization. To make a comparison between the control and antiserum-treated groups with regard to their relative susceptibilities to undergo desensitization it was necessary to select starting conditions that allowed some small amount of desensitization to be observed in the controls. For most normal cells this required application of a train of long-duration iontophoretic carb pulses (500 to 700 ms) of a few nanoamperes at 1 Hz. Some normal cells did not become desensitized even with 700-ms iontophoretic pulses, and in these cases the iontophoretic current was increased to produce some desensitization. Figure 6 shows a typical train of depolarizations obtained by applying 700-ms carb pulses to a frog muscle postjunctional membrane. The initial responses were potentiated and then as desensitization developed they declined to a steady-state level in 10 to 20 s. The parameters we used to express the amount of desensitization are given in Fig. 6. The rate of development of desensitization is expressed in terms of the time (T& required for the pulse amplitude to decrease to (AV,,, + AV3/2, where AV,,, is the maximum pulse depolarization and AVf is the depolarization pulse at steady state. The percentage desensitization (D%) = (Avm,, - AV, x lOO)lAV,,,. In paired experiments our results showed that application of undiluted

ANIMAL

MODELS

OF MYASTHENIA

GRAVIS

19

anti-AChR antiserum via microperfusion caused a significant increase in the rate of onset of receptor desensitization expressed in terms of the time taken to achieve half the maximum amount of desensitization (T~,~ = 8.9 f 4.5 s in control and 3.5 2 4.5 s in treated animals) and in the degree of desensitization as determined from the percentage decrease in iontophoretically produced depolarizations (18.8 t 6.9 to 70.3 + 8.4%). Such treated muscles in nonpaired experiments showed a 59.4% mean decrease in their response to carb, with a mean 71,2 of 15.4 s versus a 28.7% mean 71/2 of 9.6 s for the normal serum-treated controls (Table 8). The latter two 7112values are not significantly different according to a t test. A typical train of depolarizations obtained with application of 700-ms iontophoretic carb pulses to antiserum-treated frog muscle is shown in Fig. 7. The early potentiation seen in the control was lost, and an enhanced rate of desensitization was observed. The susceptibility to desensitization became further accentuated after 30 min of perfusion with antiserum (Fig. 7B). The oscillogram in Fig. 7C shows a rapid desensitization and subsequent recovery, both obtained during continuous perfusion of a fiber with anti-AChR antiserum. The recovery from desensitization monitored at 0.1 Hz following the I-Hz train of carb pulses was fairly rapid (seconds), even though the preceeding desensitization was intense judging from the fact that the depolarizations had decremented to barely perceptible ripples on the oscillogram (Fig. 7C). This recovery provided us with the assurance that the muscle had not moved appreciably during the perfusion. When the preparation was washed 30 min with Ringer’s some recovery of the cell’s chemosensitivity and some reversal of its capacity for desensitization occurred. DISCUSSION The immunization of white New Zealand rabbits with AChR protein from either electric eel or Torpedo californica was very effective in consistently producing a condition which resembled myasthenia gravis, a neuromuscular transmission deficit found in humans. Different batches of the antigen from either eel or torpedo, used on animals of different species, all produced severe myasthenia, differing only in the rate of onset. In rabbits immunized with either eel or torpedo receptor the rate of onset of myasthenia after the second immunization was fast [4 to 6 days (19)], in the rat it was slower [2 to 3 weeks; Niemi, unpublished observation, and (22)], and it was very slow in the frog [3 months (13, 16)]. This could be due to differences either in the response rate of the different immune systems or in species ability to compensate for the loss of functional AChR in the postsynaptic membrane. The almost universal myasthenic response to AChR immunization is

20

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ET AL.

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MODELS

OF MYASTHENIA

GRAVIS

21

indicative of the phylogenetic conservativeness of the AChR. The AChR seems to show a significant cross-species reactivity, even between animal species only distantly related. Heterologous AChR is capable of inducing the production of antibodies which possess the ability to react with and impair functioning of the immunized animal’s self-AChR. The consistent production of myasthenia from animal to animal and from antigen batch to antigen batch would indicate that there are a large number of functional antigenic determinants on the AChR common to all the species so far immunized. Alternatively, there may be only a few determinants on the receptor molecule which are associated with the control of ion permeability changes, and these may be very immunogenic. It is also likely that some of the determinants held in common by the eel and torpedo AChR are not necessarily those involved with ion channel control. Antisera produced in rabbits against purified torpedo AChR do not always inhibit synaptic transmission in intact eel electroplaques (lo), although these antisera are obtained from rabbits that have severe myasthenia. We always found some inhibition of synaptic transmission in the eel electroplaque after it was exposed to either rabbit anti-Torpedo antiserum or anti-eel antiserum obtained from myasthenic rabbits (22). The state of the antigen at the time of immunization may be important. Its state could control which determinants will be exposed and thus which antibodies are produced. So far, no two laboratories are producing purified AChR in the same way, and this may explain some of the differences observed. In addition, myasthenia in the rabbit may involve other factors which come into play subsequent to antibody binding, such as complement, and therefore complement-binding antibody may be the more “harmful” one. If the antibodies produced during a particular immunization are directed against determinants outside the ACh binding site of AChR they may or may not inhibit receptor function when passively applied to an in vitro preparation, depending on whether receptor conformational change is interfered with or not. Having multiple classes of antibodies could also explain the lack of correlation that is found between the level of antibody titers and the degree of physiological impairment seen in the rabbit. Myasthenic symptoms might be better correlated with a particular subclass of antibodies. When those particular fragments of the AChR that are associated FIG. 6. A typical example of receptor desensitization produced at a normal frog end-plate by 700-ms iontophoretic carbamylcholine pulses at 1 Hz using a current of 5 nA. The half-time for desensitization (T& is the time required to achieve a pulse amplitude which is one-half the maximum produced early in the train. The steady-state level of desensitization and time to achieve it is denoted in the figure by VI and 7ss.Calibration: 1.25 mV/division; lower trace, 12.5 r&division; both traces, 5 s/division.

22

NIEMI ET AL. TABLE

8

Desensitization in Frog Muscle before and after Exposure to Antireceptor Rabbit Antiserum Measured with Carbamylcholine Iontophoresis” Pulse duration and current Muscle fiber

Perfusion solution

(ms)

(nA)

Percentage AVmax (mV) desensitization

T112 (s)

Y;

A. Nonpaired Experiments 1 2 3 4

Ringer’s Ringer’s Ringer’s Ringer’s

5 6 7 8

Normal Normal Normal Normal

9 10 11 12 13

serum serum serum serum

Antiserum Antiserum Antiserum Antiserum Antiserum

Tb T T E E

700 700 500 500

12.5 12.5 37.5 15

2.5 2.0 8.0 5.5

43.6 33.1 16.0 31.3 31 + 11.4

6.3 11.3 17.1 12.7 11.9 f 4.5

7.7 23.8 24.6 21.3 19.4 + 7.9

500 500 500 500

10 25 10 10

1.3 10.9 4.8 5.8

18.7 31.4 12.2 52.6 28.7 2 17.8

3.3 12.8 15.3 6.8 9.6 k 5.5

8.1 2.7 24.7 20.8 14.1 t 10.4

508 700 700 10 300

12.5 12.5 25 180 6

2.7 3.9 2.2 1.5 2.8

59.8 60.1 75.2 50.8 61 59.4 f 10.2

14.6 40.1 44.7 12.8 7.2 12.5 3.1 10.9 7.4 10.7. 15.4 r 16.7 17.4 r 12

B. Paired Experiments 1

Ringer’s Antiserum T

200 200

6 6

12.6 3.6

27.6 51.5

20.4 3.0

26.5 10.9

2

Ringer’s Antiserum T

700 700

12.5 22.5

2.1 5.2

30.8 85.6

11.6 5.9

19.5 27.5

3

Ringer’s Antiserum E

700 700

3 6

3.3 1.7

0” 83.3

-

-

2.9

17.9

Ringer’s Antiserum T

300 300

12.5 25

5.7 1.8

16.9 60.7

3.7 2.3

10.6 14.0

4

a For definitions of percentage desensitization, rl,s, rsp, and AV,,, see Fig. 6. b Rabbit antiserum against the eel AChR is designated in the table as Antiserum E and that against torpedo AChR is designated as Antiserum T. c This value was obtained at 0.5 Hz. All other values were obtained at 1 Hz.

with AChR gating function are identified, they could be used in radioimmunoassays to determine the antibody titer against “functional” determinants which may be the more relevant titer.

ANIMAL

MODELS

OF MYASTHENIA

GRAVIS

23

FIG. 7. A typical train of depolarizations from a frog end-plate that was previously perfused with rabbit anti-AChR antiserum. A-response of frog end-plate to carbamylcholine applied iontophoretically at 1 Hz after 20 min of antiserum perfusion; pulse duration, 500 ms; current, 1.25 nA. B-after 30 min of perfusion, same as A. C-same as B but 2-nA current used. Calibrations: A-O.625 mV, 5 s/division; B and C-l.25 mV, 5 s/division.

Another problem concerns the temporal relationship that may exist between the circulating antibody level and exhibition of myasthenia. A high antibody titer without functional deficit in some individuals may mean either that these antibodies are harmless to the individual or that they precede the physiological deficits in time and indirectly contribute to the development of myasthenia. If the former is true then one might be able to

24

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ET AL.

use the “nonfunctional determinants” as tolerogens. The question of which antibodies are “harmless:’ is an open question until it can be shown that they do not indirectly affect receptor turnover or function. Correlation between serum levels of antibody and the severity of myasthenia seen in the immunized animal could also be due in part to the assay used. Purified AChR is used for the immunization and for the titer assay, but the myasthenic muscle membrane exposes only a fraction of all the antibody-binding sites that are found in the solubilized and purified AChR. It is the fraction of antibody which reacts with exposed antigenic determinants of muscle AChR which would probably better correlate with development of myasthenia. Another complication with the conventional radioimmunoassay using toxin-labeled AChR is that it precludes one from measuring the level of antibody directed against the acetylcholine binding site which is very close, if not identical to, the toxin binding site. Even using lz51-labeled antibody in the assay would not tell one the amount of AChsite-directed antibody. It is interesting that the application of rabbit anti-AChR antiserum to in vitro frog muscle or electroplaque preparations, even for prolonged periods, could not produce complete receptor block. In the intercostal muscle of some immunized animals there were many junctions that were completely insensitive to either applied acetylcholine or nerve stimulation. In all myasthenic animals it was difficult to find chemosensitive hot spots in their muscles using iontophoresis. In rabbits D and K, for example, in the biopsied samples tested, only one hot spot was found in each bundle of biopsied intercostal muscle of approximately 100 fibers, and these sensitivities ranged from 100 to 200 mV/nC. MEPPs were also not found in many junctions. Some of these MEPP-free junctions, however, were found to be sensitive to iontophoretically applied carbamylcholine, though large iontophoretic currents were required to produce depolarization. In those instances the participation of extrajunctional receptors in the depolarization cannot be ruled out. The necessity to use large iontophoretic currents to produce depolarization most probably reflects a low AChR density in the postsynaptic membrane of the AChR-immunized animals. The absence of MEPPs in muscle fibers having some remaining postjunctional membrane sensitivity to carb could also be the result of morphological disturbances, such as an increased synaptic gap distance or a shift in the juxtaposition between release sites and the so-called postjunctional “hot spots” of chemosensitivity. Functional denervation as the result of the inflammatory response (3) may have occurred at some junctions causing the absence of MEPPs and resulting in the production of spontaneous action potentials occasionally seen in some rabbit muscles. All the immunized rabbits had circulating antibodies in their blood prior to or at the time they were experiencing severe myasthenia. Serum from

ANIMAL

MODELS

OF MYASTHENIA

GRAVIS

25

these animals when placed on a normal frog muscle produced electrophysiological deficits resulting in a failure of neuromuscular transmission. This passive transfer of a neuromuscular block could be achieved with serum that had been heat treated 30 min at 56°C and therefore serum complement is not necessary for the production of neuromuscular block. It thus appears that the antibody binding, in the absence of cells or complement, can produce a deficit which could result in myasthenia but does not rule out the possibility that complement, particularly C3, could potentiate the deficit (2). In addition, antibody binding could also trigger a further reduction of AChR by involving other components of the immune system, e.g., T lymphocytes, macrophages, etc., or by accelerating receptor turnover (9). Thus immediate inhibition of AChR function in vitro may not reflect the ultimate damage a particular antibody molecule is capable of producing in the immunized animal or the myasthenic patient. Attempts to use various subunits of the receptor as the antigen or SDSdissociated AChR in place of the whole AChR molecule to produce experimental autoimmune myasthenia have failed (29). The possibility exists that the antibody might have to bridge two subunits of the native AChR to compromise its functioning and this would require that native AChR be used for the antigen. AChR subjected to purification and dissociation may not be immunologically capable of inducing antibodies with the ability to bind with two separate polypeptide chains brought close together as the result of complex folding and whose movements are associated with postjunctional ion permeability changes. Experiments are in progress to determine whether or not altered AChR could act as a tolerogen and prevent the myasthenic response from being produced by subsequent immunizations with native AChR. Another characteristic that we examined in vitro in frog muscle exposed to rabbit anti-AChR antiserum was desensitization (11, 15). We found that the PJM, after exposure to serum from immunized animals underwent a faster rate of desensitization. Normal neuromuscular junctions also undergo receptor desensitization during prolonged exposures to agonists, but not at the rate found with the anti-AChR antiserum-treated junctions. It was suggested, based on the response to intraarterial injections of ACh, that desensitization may be enhanced in the myasthenic patient (5). In support of this we found that the addition of antibody to in vitro frog muscles caused their end-plates to desensitize more readily than untreated muscles. Thus enhanced desensitization could contribute to an already existing neuromuscular transmission deficit. This could explain why anticholinesterases in some myasthenic patients aggravate instead of alleviate their condition. Thus both processes may be occurring simultaneously during a muscle’s exposure to anti-AChR antibodies, i.e., the outright inhibition of some receptors and the subtle modulation of others.

26

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The fact that some antibodies do not inhibit neuromuscular transmission upon binding (10) is significant. This could provide researchers with a tool to distinguish the more “functionally important” sites from those that are less directly involved with channel gating. REFERENCES 1. CHANG, H. W. 1974. Purification and characterization of acetylcholine receptor-l from Electrophorous electricus. Proc. Natl. Acad. Sci. U.S.A. 71: 2113-2117. 2. ENGEL, A. G., E. H. LAMBERT, AND F. HOWARD. 1977. Immune complexes (IgG and C3) at the motor endplate in myasthenia gravis. Mayo Clinic Proc. 52: 267-280. 3. ENGEL, A. G., J. M. LINDSTROM, E. H. LAMBERT, AND V. A. LENNON. 1977. Ultrastructural localization of the acetylcholine receptor in myasthenia gravis and in its experimental autoimmune model. Neurology (Miwzeapolis) 27: 307-315. 4. ENGEL, A. G., M. TSUJIHATA, J. M. LINDSTROM, AND V. A. LENNON. 1976. The motor endplate in myasthenia gravis and in experimental autoimmune myasthenia gravis. A quantitative ultrastructural study. Ann. N. Y. Acad. Sci. 274: 60-79. 5. GROB, D., AND T. NAMBA. 1976. Characteristics and mechanisms of neuromuscular block in myasthenia gravis. Ann. N. Y. Acad. Sci. 274: 143- 173. 6. GOLDSTEIN, G., AND S. WHITTINGHAM. 1966. Experimental autoimmune thymitis. An animal model of myasthenia gravis. Lancet 2: 315-318. 7. GREEN, D. P. L., R. MILEDI, AND A. VINCENT. 1975. Neuromuscular transmission after immunization against acetylcholine receptors. Proc. R. Sot. Lond. (Biol.) 189: 5768. 8. HEILBRONN, E., C. MATTSSON, L. E. THORNELL, M. SJOSTROM, E. STALBERG, P. HILTON-BROWN, AND D. ELMQVIST. 1976. Experimental myasthenia in rabbits: biochemical, immunological, electrophysiological and morphological aspects. Ann. N. Y. Acad. Sci. 274: 337-353. 9. KAO, I., AND D. B. DRACHMAN. 1977. Myasthenic immunoglobulin accelerates ACh receptor degradation. Neurology (Minneapolis) 27: 364-365. 10. KARLIN, A., E. HOLTZMAN, R. VALDERRAMA, V. DAMLE, K. Hsu, AND F. REYES. 1978. Binding of antibodies to acetylcholine receptors in Electrophorous and Torpedo electroplax membranes. J. Cell. Biol. 76: 577-592. 11. KATZ, B., AND S. THESLEFF. 1957. A study of the desensitization produced by acetylcholine at the motor endplate. J. Physiol. (London) 138: 63-80. 12. LENNON; V. A., J. M. LINDSTROM, AND M. E. SEYBOLD. 1976. Experimental autoimmune myasthenia gravis: cellular and humoral immune responses. Ann. N. Y. Acad. Sci. 274: 283-299. 13. NASTUK, W. L., W. D. NIEMI, J. T. ALEXANDER, H. W. CHANG, AND M. A. NASTUK. 1977. Myasthenia in frogs immunized against cholinergic receptor. Proc. Int. Union Physiol. Sci. Paris 13: 544. 14. NASTUK, W. L., 0. J. PLESCIA, AND K. E. OSSERMAN. 1960. Changes in serum complement activity in patients with myasthenia gravis. Proc. Sot. Exp. Biol. Med. 105: 177- 184. 15. NASTUK, W. L., AND C. H. WOLFSON. 1976. Cholinergic receptor desensitization. Ann. N.Y. Acad. Sci. 274: 130-139. 16. NIEMI, W. D., W. L. NASTUK, J. T. ALEXANDER, H. W. CHANG, AND A. S. PENN. 1978. An amphibian model for myasthenia gravis. Fed. Proc. 37: 577. 17. PATRICK, J., AND J. LINDSTROM. 1973. Autoimmune response to acetylcholine receptor. Science 180: 871-872.

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18. PATRICK, J., J. M. LINDSTROM, B. CULP, AND J. MCMILLAN. 1973. Studies on purified eel AChR and anti-acetylcholine receptor antibody. Proc. Natl. Acad. Sci. U.S.A. 70: 3334-3338. 19. PENN, A. S., H. W. CHANG, R. E. LOVELACE, W. D. NIEMI, AND A. MIRANDA. 1976. Antibodies to acetylcholine receptors in rabbits: immunochemical and electrophysiological studies. Ann. N. Y. Acad. Sci. 274: 354-376. 20. PLESCIA, 0. J., W. L. NASTUK, AND V. JOHNSON. 1969. Immune response in rabbits to homologous cardiac muscle antigens. Pages 27-32 in N. R. ROSE AND F. MILGROM, Eds., International Convocation on Immunology. Karger, BaseVNew York. 21. RICHMAN, D. P., J. PATRICK, AND B. G. W. ARNASON. 1976. Cellular immunity in myasthenia gravis in response to purified acetylcholine receptor and autologous thymocytes. N. Eng. J. Med. 2%: 694-698. 22. SANDERS, D. B., L. S. SCHLEIFER, M. E. ELDEFRAWI, N. L. NORCROSS,AND E. E. COBB. 1976. An immunologically induced defect of neuromuscular transmission in rats and rabbits. Ann. N. Y. Acad. Sci. 274: 319-336. 23. SCHOFFENIELS, E. 1957. An isolated single electroplax preparation. Improved preparation for studying ion flux. Biochim. Biophys. Acta 26: 5855%. 24. SIMPSON, J. A. 1960. Myasthenia gravis: a new hypothesis. Scot. Med. J. 5: 419-436. 25. SUGIYAMA, H., P. BENDA, J. C. MEUNIER, AND J. P. CHANGEUX. 1973. Immunological characterization of the cholinergic receptor protein from Electrophorous electricus. FEBS Lett. 35: 124-128. 26. TARRAB-HAZDAI, R., A. AHARONOV, 0. ABRAMSKY, I. YAAR, AND S. FUCHS. 1975. Passive transfer of experimental autoimmune myasthenia by lymph node cells in inbred guinea pigs. J. Exp. Med. 142: 785-792. 27. TOYKA, K. V., D. B. DRACHMAN, A. PESTRONK, AND I. KAO. 1975. Myasthenia gravis: passive transfer from man to mouse. Science 190: 397-399. 28. TOYKA, K. V., D. B. DRACHMAN, D. E. GRIFFIN, A. PESTRONK, J. A. WINKELSTEIN, K. H. FISCHBECK, JR., AND I. KAO. 1977. Study of humoral immune mechanisms by passive transfer to mice. N. Eng. J. Med. 2%: 125-131. 29. VALDERRAMA, R., C. L. WEILL, M. MCNAMEE, AND A. KARLIN. 1976. Isolation and properties of acetylcholine receptors from Electrophorus and Torpedo. Ann. N. Y. Acad. Sci. 274: 108-115. 30. WEBB, G., D. FARQUHARSON, B. HAMRELL, AND W. D. NIEMI. 1973. Studies on the blood chemistry of Electrophorous electricus and a new physiological saline solution based thereon. Biochim. Biophys. Acta 297: 313-316.