Immunization of rabbits, rats and guinea pigs with nicotinic
acetylcholine receptor from the electric organ of T. marmorata
leads to a production of antibodies in these animais. Simultaneously
a peripheral paralysis is seen, particularly .pronounced in rabbits.
In rats the symptoms are transient. The antibody titres in rabbits
are correlated to the visible symptoms. The highest antibody titres
are found in the most affected animals. Electrophysiolog;ical,
immunological and morphological analysis indicate disturbances of
the neuromuscular function, probably on the post.synaptic site.
Thymus reactions may also be involved as a thymit has been found
in the immunized rabbits

Ch. Mattsson

E. Heilbronn

English

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CROATICA CHEMICA ACTA CCACAA 47 (3) 489-495 (1975) CCA-895 YU ISSN 0011-1643 577.15 Conference Paper Immunization of Rabbits with Purified Nicotinic Acetylcholine Receptor Ch. Mattsson and E. Heilbronn Section of Biochemistry, National Defence Research Institute, Dept. 4, S-172 04 Su;ndbyberg, Sweden Immunization of rabbits, rats and guinea pigs with nicotinic acetylcholine receptor from the electric organ of T. marmorata leads to a production of antibodies in these animais. Simultaneously a peripheral paralysis is seen, particularly .pronounced in rabbits. In rats the symptoms are transient. The antibody titres in rabbits are correlated to the visible symptoms. The highest antibody titres are found in the most affected animals. Electrophysiolog;ical, immunological and morphological analysis indicate disturbances of the neuromuscular function, probably on the post.synaptic site. Thymus reactions may also be involved as a thymit has been found in the immunized rabbits. Nicotinic acetylcholine receptor (nAChR) from the electric organ of Tor-pedo marmorata was purified using affinity chromatography1,2 and shown to be a glycoprotein3. The predominant carbohydrate is mannose. Polyacrylarnide gel electroiphoresis shows two proteins with apparent molecular weights of 295 000 and 410 000 (nAChR1 resp. nAChRn). Both proteins bind [3H]-acetyl a-neurotoxin from Naja naja siamensis. Injection into rabbits of small amounts of nAChR produces symptoms of muscle weakness in the animals, visible to the eye shortly after the appearance of circulating antibodies to nAChR in the rabbit serum, and increasing in severity as the antibody titre increases4 ; 27 out of 27 animals injected have produced such symptoms. Repetitive stimulation of the peroneal nerve shows marked fatigue in muscle response, the decrement being higher in animals with higher antibody titre. Single fibre EMG, allowing measurements of the v~riability, »jitter« in neuromuscular tmnsmission time in single motor end plates showed increased jitter in all examined animals. METHODS At one week interval rabbits were injected into the thigh with 0.3 mg nAChR mixed with complete Freunds adjuvant. The animals were bled weekly and the antibody titres were determined with rocket-immunoelectrophoresis or with the indi-rect hemaglutination test5• The reciprocal of the highest serum dilution giving agglutination was recorded as the titre. Immunoglobulins were concentrated by precipitation with ammonium 'sulphate as described by Harboe and Ingild6• Fractio-nation of immunoglobulins on Sephadex G-200 was performed according to Flodin and Killander' using 0.05 M tris buffer, pH=8.0 containing 0.146 M NaCL The protein distribution was recorded at 280 nm with an Uvicord. The fractions were con-centrated and tested immunoelectrophoretically (2 h, 10 V /cm) on agarose gels made 20/o wHh respect to antiserum. 0.4-4 µg antigen was used in the electrophoresis. 490 CH. MATTSSON AND E. HEILBRONN For neurophysiological studies a .Disa electromyograph with stimulator unit was used. Stimulation was made with a bipolar electrode over the peroneal nerve at the level of the fibular head. Stimulus strength was 500/o above the stimulus for maximal muscle response. Stimulus frequence was 2, 5 and 10 impulses/s. For the two lowest frequences, 5-7 discharges were given. Initial amplitude of the response and relative amplitude decrease from the first to the fourth response was measured. The tests were repeated several times with 2-5 minutes between each. The variability (the jitter) in the neuromuscular transmission time in single motor endplates was measured by single fibre EMG as decr.ibed by Ekstedt and Stii.lberg8 • Intracellular recording of the miniature end plate potentials (MEPS) in intercostal muscle from immunized rabbits has been done using a method described by Elmquist et al.9• Autopsy of experimental animals was done and the following organs were examined by light microscopy; spleen, lung, heart, Liver, muscles from legs and diaphragma, nerves from the legs. Light as well as EM microscopy were done on the motor endplate. Acetylcholinesterase (AChE) in neuromuscular junctions was stained according to Koelle10 for comparison with normal junctions. In collaboration with dr. F. Foldes, Department of Anesthesiology, Montefiore Hospital, New York, USA, rats and guinea pigs have been injected with 0.3 mg nAChR/kg. The diaphragm and m. gastroCTlemius have been stimulated for 90 s at rate of 2 Hz in order to examine the curare sensitivity in these muscles. RESULTS Rabbits injected with isolated nicotinic 1acetylcholine receptor mixed with Freunds' complete adjuvant developed antibodies after 2 injections of receptor protein and after three injections the same gradually increasing flaccid pa;ralysis as described earlier2,4 , was seen. If not treated, death from respirntory paralysis occurs 7-14 days after the first visible symptoms. Neostigmine or Tensilon give a general, but transient relief, Tensilon having a dramatic effect. None of the control animals injected with AChE from Torpedo marmorata or with Naja naja siamensis u-neurotoxin in relevant concentrations showed any symp-toms of muscle weakness, and antibodies against Naja naja siamensis neuro-toxin11 (kindly given to us by dr. S . Fuchs), do not crossreact with the purified receptor protein. Neurotoxin or AChE impurities as the cause of the observed symptoms are therefore unlikely. The antiibody titres have been followed continuously in 5 animals and cor-related to visible symptoms. Fig. 1 shows the gradually increasing antibody 12 "~ E 10 O'I E ...... ~ ~ .... 0::: .c u <( c I :.;:: c <( 10 20 30 Time/d Fig. 1. Immunoresponse in serum of a rabbit injected with purified nAChR. t t ime of injection (i. m.). IMMUNIZATION WITH ACETYLCHOLINE RECEPTOR 491 titre in one animal. In the examined animals the antibody titre varied between 2 500-3 500 µg/ml when the first symptoms were seen and 10 000-15 000 µg/ml when the animals were killed. These titres are higher than those quoted by Patrick et al.12 and Sugiy~ma et al.13• However, Harboe and Ingild6 have shown that antibody titres fohrtd by methods where antigen and antiibody are allowed to react in free solution are 2-10 times lower than those found with techniques using reactions in a gel. Precipitation in free solutions was used by the authors quoted above. Repetitive stimulation of the peroneal nerve of rabbits shows successive fall in the amplitude of the compound muscle, action po-tential4 (Fig. 2). There is a relation between the antibody titre and the evoked muscle response, the decrement being higher as the antibody titres increase. Antibodies are always detected before the decrement. After Tensilon treatment the initial amplitude is increased and no reduction in response amplitude is seen on repetitive stimulation at the different frequencies. The conduction time from the stimulus site over the tibial anterior muscle is the same (1.4 µs) in control and sick animals and the nerve myelin sheat is normal in immunized animals. Fig. 2. Muscle action potentials recorded from tibialis anterior· muscle of the immunized rabbit at nerve stimulation frequencies 5 Hz. Single fiber EMG allows measurements of the variability in neuro-muscular transmission time in single motor endplates. The jitter was 10-30 µs in normal rabbits and was due to variability in the synaptic delays of the two motor endplates involved (F1ig. 3) . In immunized rabbits there was a marked increase in the jitter (> 100 µs) and in some of the endplates partial impulse blockade was observed (Mattsson, Hilton-Br.own, Stalber,g and Heilbronn, to be published). The mirl~ature endplate potentials (MEPS) in the intercostal muscle of healthy and experimental rabbits have been registered. The MEPS amplitude was found to be markedly decreased in the exper·imental animals. Only 10-200/o of the normal value was found in all the examined animals (Fig. 4) (Elmqvist, Libelius, Mattsson and Heilbrnnn, to be published). The antiserum and the isolated immunoglobulin fraction precipitated recep-tor protein, as measured by the [3H]-acetyl-a-neurotoxin binding capacity of 492 CH. MATTSSON AND E. HEILBRONN Fig. 3. Left : Increased pa t holog ical jitter in a n immunized rabbit. Right : Normal jitter in a control rabbit. l mV _\ \ ( I J b'.r! '··· r ' ' . Fig. 4. MEPS amplitude in a normal rabbit intercostal muscle (above) and in an immunized rabbit (below). lo c: x 2 0 :; ., c I I 0.5 M -a c :;J 0 ..0 0 E a. 50 150 J.il antiserum Fig. 5. Precipitation of purified nAChR b y anti-nAChR from rabbit as meas ured by the [' HJ acetyl-a.-neurotoxin binding capacity of the supernatant. lMM lJN'IZ A T I ON' WITH ACETYLCHOLINE RECEPTOR 493 Fig. 6. Rocket immunoelectrophoresis o f nAChR on an 10/o agarose gel made ZO/n with respect to anti-nAChR. Wells from left: 1-3 nAChR 3.6, 1.8 and 0.9 µg resp. 4. isolated nAChR1• 5. isolate d nAChRn. 10 12 11. 16 18 20 22 21. 26 28 30 Fract ion Nr Fig. 7. Gel filtration of rabbit a-globulin on Sephadex G-200. 494 CH. MA TT SS ON AND E. HEILBRONN the solution, from a Triton X-100 extract of Tor pedo electric organ as well as from a solution of purified rece,ptor protein (Fig. 5). In immunoelectrophoresis the receptor protein reacted with the antibodies in one single line (Fig. 6). The two proteins isolated by polyacrylamide gel electrophoresis also reacted in one single line indicating that they are immunologically identical. It was not possible to adsorb antibodies from rabbit serum by mixing serum with freeze dried Torpedo electric organ suggesting that the immunologically active part of the molecule is buried within the membrane. In order to study which lg class of the rabbit antiserum is responsible for the antibody activity rabbit y-globulin was fractionated on a G-200 column (Fig. 7) . The antibody activity was determined with immunoelectrophoresis. Only the second peak precipitated the antigen suggesting mainly receptor antibodies of the IgG type in the serum. In collaboration with dr. Aarli from the Department of Neurology, University of Bergen, Norway, the receptor protein has been crosstested ,against several sera from myastenic patients using an indirect haemagglutination test. No positive results were obtained. No pathological signs were found in the spleen, lung, kidney, adrenal glands, liver and heart of the immunized rabbits. Muscle · threads are oc-casionally atrophic or slightly inflamed, but no pronounced myocyte is observed. In some animals a relatively large number of eosinophilic granulocytes is seen among the lymphoid cells, possibly indicating a thymit. EM-microscopy on the endplate region indicates changes, the folding of the endplate region is reduced. Four receptor injected rats were compared with 4 others rats injected with the same amount of serum a}bumin and with 4 uninj ected rats. The 4 receptor injected rats exhibited greatly increased curare sensitivity which was more marked in the diaphragm than in the gastrocnemius. The one guinea pig tested so far was also more sensitive to curare than the controls. REFERENCES 1. E. Kar 1 s son, E. He i 1 bro n n, and L. W id 1 u n d, FEBS Lett. 28 (1972) 107. 2. E. He i 1 bro n n and Ch. Mattsson, J. Neurochem. 22 (1974) 315. 3. Ch. Mattsson and E. He i 1 bro n n, J. Neurochem. 25 (1975), in press. 4. E. He i b r on n, Ch. Matts s o n, S. S t a 1 b e r g, and P. Hi 1 to n -- Brown, J. Neurol. Sci. 24 (1975) 59. 5. J. A. A a r 1 i, Clin. Exp. Immunol. 10 (1972) 453. 6. Harb o en and A. Ing i 1 d, Immunization, isolation of immunoglobulins, esti-mation of antibody titre in immunology, in: N. H. Axe 1 sen, J. Kr o 11, and B. Week e (Eds.), A Manual of quantitative Immunoelectrophoresis, Methods and Applications Vol. 2, Supp. 1, Universitetsfiirlagt, Oslo 1973, pp. 161-164. 7. P. F 1 o din and J . Ki 11 and er, Biochim. Biophys. Acta 63 (1962) 403. · 8. J. Ek st e d t and E. St a 1 berg, Electroencephalog. Clin. Neurophysiol. 27 (1969) 557. 9. D. E 1 m qui st, W. W. Hofmann, I. Ku g e 1 berg, and D. M. J. Qua-s tel, J. Physiol. 174 (1964) 417. 10. G. B. Koe 11 e, J . Pharmacol. Exp. Ther. 103 (1951) 153. 11. A. Ah a r on o v, D. Gur a r i, and S. Fuchs, Eur. J. Biochem. 45 (1974) 297. 12. J. Patric, J. Lindstrom, B. Cu 1 p, and J. Mc Mi 11 an, Proc. Nat. Acad. Sci. U .S.A. 70 (1973) 3334. 13. M. Sugiyama, P. Benda, J.-C. Meunier, and J.-P. Changeux, FEBS Lett. 35 (1973) 124. IMMUNIZATION WITH ACETYLCHOLINE RECEPTOR 495 DISCUSSION W. N. Aldridge: Do you consider the symptoms in the animal can be explained by the reduction of effective receptor molecules (shown by neurotoxin binding)? R. D. O'Brien: Why did you select intercostal muscles for a study of toxin binding? The animal is surviving well, so the intercostals are clearly in good order; why not study the leg muscles in which paralysis is evident? M. E. Eldefrawi: Your data on the binding of [3H]-neurotoxin to muscles from normal immunized rabbits showed only 200/o reduction iJn number of receptor si,tes. Do you think that such a reduction could explain the paralysis and death of the 'immunized rabbits and how do you compare your data wiih dr. Barnard's published v(l.lues of at least 500/o excess receptor (or spare receptor) found in rat diaphragms? Ch. Mattsson: The questions of Drs. Aldddge, O'Brien and Eldefrawi could be answered together. Our measurements were done on the intercostal muscles because MEPS were measured cm this tissue and we wanted to be a:ble to correlate the two. The animals were living and breathing when this was done, thus no complete block of neurotransmission there could be expected. Skeletal muscles, e. g. of the legs, might have shown greater block of receptors. In any case, Albuquerque et. al. (Life Sci. 12 (1973) 545) have shown that block of 250/o of receptors impairs neurotransmission. Whether the reduction in function al receptors is the whole truth or not i's too early to say. Certainly it seems to be an important part of the 1paralysis and the rabbits showed normal conduction in their motor nerves. I. Silman: What kind of symptoms do you see in AChR-immunized rats? Have you done EMG measurements on them? Ch. Mattsson: No EMG has so far been done on r ats. Only vtsible symptoms have been observed. These symptoms are maximal after about 3 weeks and not so pronounced as in rabbits. However, in some experiments done together with dr. Foldes (Mon-tefiori Hospital, New York), increased curare sensitivity was shown in these animals, particularly in the diaphragm. B. StamenoviC: (a) Regarding the reduced amplitude of the MEPS in the immunized rabbits, what was the resting membrane potential of the impaired muscle fibres during the recording of MEPS? (b) When suggesting the postsynaptic change at the neuro-muscular junction for the observed decrease in MEPS amplitude, have you any evidence of the unit size of MEPS as well as of the mean quantum content of EPP in such conditions, because the presented MEPS are very like those recorded by the Elmquist group in myasthenic patients. Ch. Mattsson: (a) The resting membrane potential was 80 mV in immunized as well as in control rabbits. (b) The mean quantum content has not yet been determined, only the MEPS amplitude which was 1 mV for controls and 0.2 mV or lower for dmmunized rabbi.ts. It remains to see whether this is due to only postsynaptic effects or if presynaptic effects are involved as well. Iontophoretic application of ACh is cur-rently studied. 496 SAZETAK Imunizacija kuniea prociscenim nikotinskim acetnkolinskim receptorom Ch. Mattsson i Edith Heiibronn Imunizacija kunica, stakora i zamorca nikotinskim acetilkolinskim receptorom elektrienog organa T . marmorata, uzrokuje stvaranja antitijela u tim zivotinjama. Utvrdena je istovremena pojava periferne paralize, koja je osobito jako izrazena u kuniea dok SU u stakora simptomi prolazni. Titar antitijela u kunicu u korelaciji je s vidljivim simptomima. Elektrofizioloska, imunoloska i morfoloska analiza upucuj u na poremeeaj neuromuskularne funkcije, vjerojatno na postsinaptickoj strani. Reak-cije timusa mogle bi takoder biti ukljucene, buduci da je u imuniziranim kunicima naden jedan timit. ODJEL ZA BIOKEMIJU, ISTRA!ZIVACKI INSTITUT NARODNE ODBRANE, ODJEL 4, S-172 04 SUNDYBERG, SVEDSKA 

Immunization of Rabbits with Purified Nicotinic Acetylcholine Receptor

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