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Effect of denervation on the organization of the postsynaptic membrane of the electric organ ofTorpedo marmorata
Brain Research, 90 (1975) 133-138 {(~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
133
Short Communications
Effect of denervation on the organization of the postsynaptic membrane of the electric organ of Torpedo mormorofo
F. CLEMENTI, B. C O N T | - T R O N C O N I , D. P E L U C H E T T | AND M. M O R G U T T I
Department of Pharmacology, University of Milan and C.N.R. Center of Cytopkarmacology, Milan (Italy) (Accepted February 24th, 1975)
It is known that the presynaptic nervous input influences the organization of the muscular plasmalemma. This effect is particularly evident both during ontogenesisl0 12,14 and after denervation a,9,2z, and concerns principally two membrane proteins, namely acetylcholine receptor1,9, lz,~2 and muscular acetylcholinesterase (AchE)la,1% After denervation, a spreading of the receptor protein from the subsynaptic cleft to the entire plasmalemma has been widely reported 1,9,22, together with a slow and progressive decrease in AchE content of the muscle aa,16. Since the electric organs of Torpedo are now extensively used not only as a rich source of nicotinic receptor but also as a useful model for the study of the cholinergic synapse, we investigated whether the relationships between the innervating fibers and the postsynaptic membrane are comparable to those existing in the striated muscles of vertebrates. Postsynaptic plasma membrane was studied at different times after denervation morphologically, using the freeze fracture technique, and biochemically by assaying the AchE activity and the toxin binding properties of the electric organ. Torpedines marmoratae, kindly supplied by Dr. Martin of the Stazione Zoologica (Naples), were kept in artificial oxygenated sea water at 18-20 °C. Under light urethane (2?/o0) anesthesia the nerves to the electric organ were exposed, and the 3 caudal nerves were ligated and sectioned. All the animals recovered in a very short period. Biopsies from the denervated and normally innervated tissues were taken, under urethane anesthesia 7, 15, 30 and 60 days after denervation. The tissue samples were fixed in 2.5% gluteraldehyde buffered with 0.14 M phosphate buffer, pH 7.3. Small fragments of the fixed tissues were infiltrated with increasingly concentrated solutions of glycerol in phosphate buffer, frozen in Freon 22 cooled with liquid nitrogen, freeze fractured and replicated in a Balzer 301 apparatus provided with an electron gun, following the technique of Moor et al. 17. Ache activity and protein were assayed according to Ellman et al. s and to
134
Fig. I. Freeze fractu]ed rcplic:l or a normal clectroplax cell (E) showing the inf~crvated iilcc, l'~}c fracture exposes the nerve endings (NE), their presynaptic membrane and a snlall area of p~sb, synaptic membrane (POM). Two cross-fraclured synaptic folds (SF) are also shown. S sho~s u Schwann cell process. Inset: higher magnitication of the A and B lace of the postsynaptic membrane, In the B face the laltice of repeating subtHlits is particularly evident (arrowheads). In all the ntlclographs the circled arro~ shoves lhe direction of shadowing. 38,000: inset 140,000.
Lowry et al. ~ respectively. Tile binding of the [3H]a-toxin o f N a j a nigricollis was measured a c c o r d i n g to Cohen et al. 7 a n d corrected for b a c k g r o u n d a b s o r p t i o n on Millipore filters. Both A c h E activity and toxin binding were d e t e r m i n e d in h o m o genized electric organ 60 days after denervation. F r o m the careful studies o f Sheridan ea on thin sections, it emerges that the electric organ o f Torpedo consists o f a b o u t 500 stacks, each consisting o f n u m e r o u s and repetitive flat cells with their ventral face richly innervated by cholinergic nerve fibers originating in the electric lobes (Fig. 1). F u r t h e r studies carried out using the freeze fracture technique by Orci et al. I:) a n d by C a r t a u d 4,~' have shown in more detail the fine structure o f e l e c t r o p l a x m e m branes. In particular the postsynaptic m e m b r a n e has a peculiar substructure with a different particle c o m p o s i t i o n on the 2 c o m p l e m e n t a r y faces. The A-face has n u m e r o u s h o m o g e n o u s l y dispersed 8 9 nm-particles, as usually f o u n d in other p l a s m a mere-
135
Fig. 2. Freeze fracture of a 60-day-denervated electroplax. Between the Schwann cells (S) and the postsynaptic membrane of the electroplax cell no nerve endings are present. Rarely some residues of degenerated terminals are visible, as shown in the left upper corner. The fractured faces (A and B} of the postsynaptic membrane have a normal appeara1:ce. > 55,000.
branes, while the B-face is characterized by smaller particles arranged in an ordered lattice (Fig. 1A and B). It was originally suggested by Orci et al. 19 and later supported with further experimental evidence by Cartaud 4,5 and Nikel and Potter 18 that the small particles forming the lattice may represent the receptor protein. In our experiments we observed that presynaptic nerve fibers did not disappear completely from the electric organ until a b o u t 60 days after denervation. However, even at this late time, no further modifications were apparent in the electric organ. The general appearance of the postsynaptic membrane was normal and the postsynaptic infoldings were still present in a normal shape and n u m b e r (Figs. 2 and 3). The distribution of the particles in the A-face of the plasmalemma was very similar to that o f the non-denervated cells. N o modification appeared in number, distribution and size of the small particles present in the B-face o f plasmalemma either in the subsynaptic cleft or in the postsynaptic infoldings (Fig. 3). The normal morphological distribution o f particles in the electroplax excitable membranes was in agreement with
136
Fig. 3. Freeze fracture of a 60-das-denervated electroplax. The fracture exposes the A and B ktce t~ 2 synaptic folds. The arrowheads, on the B face, sho,~ an array of repeating particles closely ~,pz~_ed x 145,000.
137 TABLE
1
ACETYLCHOLINESTERASE ACTIVITY AND TOXIN BINDING PROPERTY OF
Torpedo ELECTRIC
ORGAN BEFORE
AND AFTER DENERVATION T h e s e values are e x p r e s s e d as the m e a n o f 2 consistent e x p e r i m e n t s .
Normal Denervated
AchE activity ( U/mg* o/protein)
, aH:'ct-toxin bDlding (nM/g of protehl)
13.6 19.0
5.65 5.51
* 1 unit will hydrolyze 1 /~M of acetylcholine/min.
the biochemical determinations of AchE activity and of the toxin binding property. The toxin binding property did not change significatively after denervation and the small increase of AchE activity was also not significant (Table I). Our morphological and biochemical data clearly show that denervation does not affect the content and distribution of 2 specific proteins in the excitable membrane of Torpedo electroplax. These data are in agreement with the observations of Rosenberg et al. 2° and those of Burgeois et al. 3, on the effect of denervation on the Electrophorus electroplax. They found a rather rapid and progressive decrease of choline acetylase while AchE activity and the toxin binding property were not affected. However, the data on fish electric organs are rather in contrast with the effects of denervation on the skeletal muscle, namely the appearance of extrajunctional cholinergic receptors ~,9,'~ and the decrease of AchE activity 13,16. This discrepancy may be explained by several considerations. It has recently been demonstrated that the extrajunctional receptors in denervated muscle have a turnover higher than the junctional receptors of normal muscles e and that only the extrajunctional receptors vary with the presence or not of innervating fibers. The turnover of membrane proteins from the Torpedo electric organ, kept in the aquarium, is very slow: much slower than that of mammalian tissues 6. This difference in turnover may alone explain the insensitivity of the receptors to the nervous input and the relative rigidity of the electroplax membrane. Furthermore, in the electroplax the excitable plasma membrane is nearly completely covered by nerve terminals, so that the membrane is normally 'saturated' by junctional receptors and it is possible that no space is available for the extrajunctional ones. Our data, and the observations reported in the literature, indicate that although fish electric organs may be useful as a model for studying the physiology and pharmacology of neuromuscular junctions, it will be necessary to take into account its possible numerous limitations. We are particularly indebted to Dr. Meunier who generously gave us the purified [aH](~-toxin. The skillful help of Franco Crippa, Paolo Tinelli and Cecilia Gotti was greatly appreciated.
13S I AXELSSC,N, J., \N!} j IllS* 1 i 1, •., A stud5 of superscnsiti~iD m denerva,ed m a m m a l i a n >i, clela! musclc, J. Pto~r~/, Lore".. 149(i959) I7S 193. 2 BEI',<, I). K...~'-q) IIAii, / . \~,., l a t e oi ~,-bungarotoxin b o u n d to acet31choline recel'q~w-, o~ n o r m a l and denevvated ntuscJc, SH~,mc, 184(1974) 473 475. 3 BouR/;i: As, J. P.. P~H,oi, .I. [... t"(~'1114, /\., AND CttAN(iL:E X, J. I~'., C o n s e q u e n c e s ot denerxation on the distribution of {he cholincrgic (nicotinicl rcceDtor sites from E/cctrophor,~ ,4,:~trict,,, revealed by high resolution autoradiograph>. Braill Research, 62 (1973) 557 563. 4 CARr.a, tH). j., ~',ructural o r g a n i z a t k m of the excitable m e m b r a n e o1' the 7orpedo IHcov)mrahi electric organ. In J. V. SA',DIRS A',.P~ D. J. (;OODCHItD (Eds.), Elccti'ou Micro~SCOl,r. l),/. IL Australian A c a d e m y of Science, ( ' a n b c r r a , 1974, pp. 284 285. 5 CaRIatq), J+, [~ENE,)t:, II, ~. L., (. OIt, N, J. [~., M,/UNIER, J ('., AND CHANI.ib.UX, J. P., Presence el a lattice structure in naembrane fragments rich in nicotinic receptor protein fiom the electric organ of Torpe~h~ marmoram, t:'t~BS LcH., 33 (1973) 109 113. 6 CLEMtNI ;, E., (ONII-'[RON('()NI, B., kN,) ~'IELDOLESI, J., umpublished resuhs. 7 COH[->,, J. B., Wll~el< M.. t h CHE~, M., ,',Ni~ (_THANGEUX,J. P., Purification from Torpe&? marmo;'ata electric tissue of iltel'rll'ralle fragments particularl.v rich in cholinergic receptor protein, F E B S L~,tt., 26 (1972) -1,3 J7. 8 E, LMA>,, G. L., ( o t m l ' , ~ , K. 1)., A ~ o J ~ s , V. Jr., AnD I-:EATERSTONE, R. M., A new a n d rapid colorimetric delerminatiot3 of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 9 EIMOvIs;, 1)., ,',vl} ]'H,SLIfl, S., A study on acetylcholine induced contractures in denervated m a n n n a l i a n muscle, ,4cla plutrmocol. (A'l~h.), 17 (1960) 84 93. 10 FAMBROU(;H, D. M., [{AI'~.IZIl_1,, H. ('., 13(}V<,LL, J. A., RASH, J. E., AND JOSI-Pft, N., On the dilllerentialion a n d organization of the surface m e m b r a n e of a postsynaptic cell. The skeletal muscle tit:el. In V. L. I-h-r,,-,H, (Ed.), ~SrnapHc I)'a,.smission and Neuronal l/tte,'a~'lion, Vo[. 28, Raven Press, New York, 1974, pp. 285 313. I1 EAMBROU(;;I, I). M.. iJAV, IZtL,L. H. ('., RASH. ,I. E., a n d RIICHIE, A. K., Receptor properties of developing muscle, An/;. \ . }. ,4cad. SH.. 228 (1974) 47 62. 12 GIAcOm"q, G., FHo~,,x~o (L, W~m!l~, M., BoOvE3, P., AXe CHANC;eAUX, .I.P., Effects of a snake ¢z-neurotoxin on the d e \ e l o p m e n t of innervated skeletal muscles in chick embryo, Proc, m;t. Acad. Sci. (ll2;.sh.,,, 70 (t973) 1708 1712. 13 HALt, Z. W., Multiple forms of acetylcholmesterase and their distribution in endplate and non endplate regions of rat d i a p h r a g m muscle, J. Neurobiol. 4 (1973) 343 361. 14 HAm~ls, A. J., Rolc of acetylchotine receptors in synapse formation. In V. L. BENNETI (Ed.), S3'mtptic Tra/tsmA,sio, *¢m/ ¢Y~'tl,Oll*l[ It;leracthm, Vet. 28, Raven Press, New York, 1974, pp. 315 337. 15 LO\VRY. O. H., ROSEBROU{Itl, N. J., VAP,R, A. L., ANt) RANDALL, R. J., Protein m e a s u r e m e n t with Fol in phenol reagent, J. /fro/. Chem., 193 (1951)265-275. 16 McCAvA>< M. W., Biochemical effects of denervation on normal a n d dystrophic muscle: acetylcholinesterase and choline acetyhransferase, l,i/i, Sci., 5 (1966) 1459-1465. 17 MOORE, H., Mi)LIilHALIR. K., WALI)NER, H., AND EREY-WYSSLING, A.. A new freezing-ultramicrotome, J. biophy.s. /~h)chem. ('.vtol., 10 (1961) I II. 18 N.('KI L, E., AN[) P o l r e r , 1,. T., Ultrastructure o f isolated m e m b r a n e s of Torpedo electric tissue, Brat, Research, 57 (1973) 508 517. 19 Orf~, L., PeRRELET, A., A,aD DUNAN1", Y., A peculiar s u b s t r u c t u r e in the postsynaptic m e m b r a n e of Torpedo electroplax, Prec. m*t. Acad. S~i. (Wash.), 71 (1974) 307-310. 20 Rose~mJ~_~, P,, MaCKF,', E. A., H~MAX', H. B., AYD DETTBARN, W. D., Choline acety[ase a n d cholinesterase activit5 in denervated electroplax, Biochim. biophys. Acre ~Amst.), 82 (1964) 266 275. 21 SHERIDAN, M. N., ] h e line structure of the electric organ of Torpedo marmorata. J. Cell Bh~l., 24 (1965) 129 141. 22 THeSt,VVF. S., Physiological effects of denervation of muscle, Attn. N . Y . Acad. Sci., 228 (1974) 89 104.
133
Short Communications
Effect of denervation on the organization of the postsynaptic membrane of the electric organ of Torpedo mormorofo
F. CLEMENTI, B. C O N T | - T R O N C O N I , D. P E L U C H E T T | AND M. M O R G U T T I
Department of Pharmacology, University of Milan and C.N.R. Center of Cytopkarmacology, Milan (Italy) (Accepted February 24th, 1975)
It is known that the presynaptic nervous input influences the organization of the muscular plasmalemma. This effect is particularly evident both during ontogenesisl0 12,14 and after denervation a,9,2z, and concerns principally two membrane proteins, namely acetylcholine receptor1,9, lz,~2 and muscular acetylcholinesterase (AchE)la,1% After denervation, a spreading of the receptor protein from the subsynaptic cleft to the entire plasmalemma has been widely reported 1,9,22, together with a slow and progressive decrease in AchE content of the muscle aa,16. Since the electric organs of Torpedo are now extensively used not only as a rich source of nicotinic receptor but also as a useful model for the study of the cholinergic synapse, we investigated whether the relationships between the innervating fibers and the postsynaptic membrane are comparable to those existing in the striated muscles of vertebrates. Postsynaptic plasma membrane was studied at different times after denervation morphologically, using the freeze fracture technique, and biochemically by assaying the AchE activity and the toxin binding properties of the electric organ. Torpedines marmoratae, kindly supplied by Dr. Martin of the Stazione Zoologica (Naples), were kept in artificial oxygenated sea water at 18-20 °C. Under light urethane (2?/o0) anesthesia the nerves to the electric organ were exposed, and the 3 caudal nerves were ligated and sectioned. All the animals recovered in a very short period. Biopsies from the denervated and normally innervated tissues were taken, under urethane anesthesia 7, 15, 30 and 60 days after denervation. The tissue samples were fixed in 2.5% gluteraldehyde buffered with 0.14 M phosphate buffer, pH 7.3. Small fragments of the fixed tissues were infiltrated with increasingly concentrated solutions of glycerol in phosphate buffer, frozen in Freon 22 cooled with liquid nitrogen, freeze fractured and replicated in a Balzer 301 apparatus provided with an electron gun, following the technique of Moor et al. 17. Ache activity and protein were assayed according to Ellman et al. s and to
134
Fig. I. Freeze fractu]ed rcplic:l or a normal clectroplax cell (E) showing the inf~crvated iilcc, l'~}c fracture exposes the nerve endings (NE), their presynaptic membrane and a snlall area of p~sb, synaptic membrane (POM). Two cross-fraclured synaptic folds (SF) are also shown. S sho~s u Schwann cell process. Inset: higher magnitication of the A and B lace of the postsynaptic membrane, In the B face the laltice of repeating subtHlits is particularly evident (arrowheads). In all the ntlclographs the circled arro~ shoves lhe direction of shadowing. 38,000: inset 140,000.
Lowry et al. ~ respectively. Tile binding of the [3H]a-toxin o f N a j a nigricollis was measured a c c o r d i n g to Cohen et al. 7 a n d corrected for b a c k g r o u n d a b s o r p t i o n on Millipore filters. Both A c h E activity and toxin binding were d e t e r m i n e d in h o m o genized electric organ 60 days after denervation. F r o m the careful studies o f Sheridan ea on thin sections, it emerges that the electric organ o f Torpedo consists o f a b o u t 500 stacks, each consisting o f n u m e r o u s and repetitive flat cells with their ventral face richly innervated by cholinergic nerve fibers originating in the electric lobes (Fig. 1). F u r t h e r studies carried out using the freeze fracture technique by Orci et al. I:) a n d by C a r t a u d 4,~' have shown in more detail the fine structure o f e l e c t r o p l a x m e m branes. In particular the postsynaptic m e m b r a n e has a peculiar substructure with a different particle c o m p o s i t i o n on the 2 c o m p l e m e n t a r y faces. The A-face has n u m e r o u s h o m o g e n o u s l y dispersed 8 9 nm-particles, as usually f o u n d in other p l a s m a mere-
135
Fig. 2. Freeze fracture of a 60-day-denervated electroplax. Between the Schwann cells (S) and the postsynaptic membrane of the electroplax cell no nerve endings are present. Rarely some residues of degenerated terminals are visible, as shown in the left upper corner. The fractured faces (A and B} of the postsynaptic membrane have a normal appeara1:ce. > 55,000.
branes, while the B-face is characterized by smaller particles arranged in an ordered lattice (Fig. 1A and B). It was originally suggested by Orci et al. 19 and later supported with further experimental evidence by Cartaud 4,5 and Nikel and Potter 18 that the small particles forming the lattice may represent the receptor protein. In our experiments we observed that presynaptic nerve fibers did not disappear completely from the electric organ until a b o u t 60 days after denervation. However, even at this late time, no further modifications were apparent in the electric organ. The general appearance of the postsynaptic membrane was normal and the postsynaptic infoldings were still present in a normal shape and n u m b e r (Figs. 2 and 3). The distribution of the particles in the A-face of the plasmalemma was very similar to that o f the non-denervated cells. N o modification appeared in number, distribution and size of the small particles present in the B-face o f plasmalemma either in the subsynaptic cleft or in the postsynaptic infoldings (Fig. 3). The normal morphological distribution o f particles in the electroplax excitable membranes was in agreement with
136
Fig. 3. Freeze fracture of a 60-das-denervated electroplax. The fracture exposes the A and B ktce t~ 2 synaptic folds. The arrowheads, on the B face, sho,~ an array of repeating particles closely ~,pz~_ed x 145,000.
137 TABLE
1
ACETYLCHOLINESTERASE ACTIVITY AND TOXIN BINDING PROPERTY OF
Torpedo ELECTRIC
ORGAN BEFORE
AND AFTER DENERVATION T h e s e values are e x p r e s s e d as the m e a n o f 2 consistent e x p e r i m e n t s .
Normal Denervated
AchE activity ( U/mg* o/protein)
, aH:'ct-toxin bDlding (nM/g of protehl)
13.6 19.0
5.65 5.51
* 1 unit will hydrolyze 1 /~M of acetylcholine/min.
the biochemical determinations of AchE activity and of the toxin binding property. The toxin binding property did not change significatively after denervation and the small increase of AchE activity was also not significant (Table I). Our morphological and biochemical data clearly show that denervation does not affect the content and distribution of 2 specific proteins in the excitable membrane of Torpedo electroplax. These data are in agreement with the observations of Rosenberg et al. 2° and those of Burgeois et al. 3, on the effect of denervation on the Electrophorus electroplax. They found a rather rapid and progressive decrease of choline acetylase while AchE activity and the toxin binding property were not affected. However, the data on fish electric organs are rather in contrast with the effects of denervation on the skeletal muscle, namely the appearance of extrajunctional cholinergic receptors ~,9,'~ and the decrease of AchE activity 13,16. This discrepancy may be explained by several considerations. It has recently been demonstrated that the extrajunctional receptors in denervated muscle have a turnover higher than the junctional receptors of normal muscles e and that only the extrajunctional receptors vary with the presence or not of innervating fibers. The turnover of membrane proteins from the Torpedo electric organ, kept in the aquarium, is very slow: much slower than that of mammalian tissues 6. This difference in turnover may alone explain the insensitivity of the receptors to the nervous input and the relative rigidity of the electroplax membrane. Furthermore, in the electroplax the excitable plasma membrane is nearly completely covered by nerve terminals, so that the membrane is normally 'saturated' by junctional receptors and it is possible that no space is available for the extrajunctional ones. Our data, and the observations reported in the literature, indicate that although fish electric organs may be useful as a model for studying the physiology and pharmacology of neuromuscular junctions, it will be necessary to take into account its possible numerous limitations. We are particularly indebted to Dr. Meunier who generously gave us the purified [aH](~-toxin. The skillful help of Franco Crippa, Paolo Tinelli and Cecilia Gotti was greatly appreciated.
13S I AXELSSC,N, J., \N!} j IllS* 1 i 1, •., A stud5 of superscnsiti~iD m denerva,ed m a m m a l i a n >i, clela! musclc, J. Pto~r~/, Lore".. 149(i959) I7S 193. 2 BEI',<, I). K...~'-q) IIAii, / . \~,., l a t e oi ~,-bungarotoxin b o u n d to acet31choline recel'q~w-, o~ n o r m a l and denevvated ntuscJc, SH~,mc, 184(1974) 473 475. 3 BouR/;i: As, J. P.. P~H,oi, .I. [... t"(~'1114, /\., AND CttAN(iL:E X, J. I~'., C o n s e q u e n c e s ot denerxation on the distribution of {he cholincrgic (nicotinicl rcceDtor sites from E/cctrophor,~ ,4,:~trict,,, revealed by high resolution autoradiograph>. Braill Research, 62 (1973) 557 563. 4 CARr.a, tH). j., ~',ructural o r g a n i z a t k m of the excitable m e m b r a n e o1' the 7orpedo IHcov)mrahi electric organ. In J. V. SA',DIRS A',.P~ D. J. (;OODCHItD (Eds.), Elccti'ou Micro~SCOl,r. l),/. IL Australian A c a d e m y of Science, ( ' a n b c r r a , 1974, pp. 284 285. 5 CaRIatq), J+, [~ENE,)t:, II, ~. L., (. OIt, N, J. [~., M,/UNIER, J ('., AND CHANI.ib.UX, J. P., Presence el a lattice structure in naembrane fragments rich in nicotinic receptor protein fiom the electric organ of Torpe~h~ marmoram, t:'t~BS LcH., 33 (1973) 109 113. 6 CLEMtNI ;, E., (ONII-'[RON('()NI, B., kN,) ~'IELDOLESI, J., umpublished resuhs. 7 COH[->,, J. B., Wll~el< M.. t h CHE~, M., ,',Ni~ (_THANGEUX,J. P., Purification from Torpe&? marmo;'ata electric tissue of iltel'rll'ralle fragments particularl.v rich in cholinergic receptor protein, F E B S L~,tt., 26 (1972) -1,3 J7. 8 E, LMA>,, G. L., ( o t m l ' , ~ , K. 1)., A ~ o J ~ s , V. Jr., AnD I-:EATERSTONE, R. M., A new a n d rapid colorimetric delerminatiot3 of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 9 EIMOvIs;, 1)., ,',vl} ]'H,SLIfl, S., A study on acetylcholine induced contractures in denervated m a n n n a l i a n muscle, ,4cla plutrmocol. (A'l~h.), 17 (1960) 84 93. 10 FAMBROU(;H, D. M., [{AI'~.IZIl_1,, H. ('., 13(}V<,LL, J. A., RASH, J. E., AND JOSI-Pft, N., On the dilllerentialion a n d organization of the surface m e m b r a n e of a postsynaptic cell. The skeletal muscle tit:el. In V. L. I-h-r,,-,H, (Ed.), ~SrnapHc I)'a,.smission and Neuronal l/tte,'a~'lion, Vo[. 28, Raven Press, New York, 1974, pp. 285 313. I1 EAMBROU(;;I, I). M.. iJAV, IZtL,L. H. ('., RASH. ,I. E., a n d RIICHIE, A. K., Receptor properties of developing muscle, An/;. \ . }. ,4cad. SH.. 228 (1974) 47 62. 12 GIAcOm"q, G., FHo~,,x~o (L, W~m!l~, M., BoOvE3, P., AXe CHANC;eAUX, .I.P., Effects of a snake ¢z-neurotoxin on the d e \ e l o p m e n t of innervated skeletal muscles in chick embryo, Proc, m;t. Acad. Sci. (ll2;.sh.,,, 70 (t973) 1708 1712. 13 HALt, Z. W., Multiple forms of acetylcholmesterase and their distribution in endplate and non endplate regions of rat d i a p h r a g m muscle, J. Neurobiol. 4 (1973) 343 361. 14 HAm~ls, A. J., Rolc of acetylchotine receptors in synapse formation. In V. L. BENNETI (Ed.), S3'mtptic Tra/tsmA,sio, *¢m/ ¢Y~'tl,Oll*l[ It;leracthm, Vet. 28, Raven Press, New York, 1974, pp. 315 337. 15 LO\VRY. O. H., ROSEBROU{Itl, N. J., VAP,R, A. L., ANt) RANDALL, R. J., Protein m e a s u r e m e n t with Fol in phenol reagent, J. /fro/. Chem., 193 (1951)265-275. 16 McCAvA>< M. W., Biochemical effects of denervation on normal a n d dystrophic muscle: acetylcholinesterase and choline acetyhransferase, l,i/i, Sci., 5 (1966) 1459-1465. 17 MOORE, H., Mi)LIilHALIR. K., WALI)NER, H., AND EREY-WYSSLING, A.. A new freezing-ultramicrotome, J. biophy.s. /~h)chem. ('.vtol., 10 (1961) I II. 18 N.('KI L, E., AN[) P o l r e r , 1,. T., Ultrastructure o f isolated m e m b r a n e s of Torpedo electric tissue, Brat, Research, 57 (1973) 508 517. 19 Orf~, L., PeRRELET, A., A,aD DUNAN1", Y., A peculiar s u b s t r u c t u r e in the postsynaptic m e m b r a n e of Torpedo electroplax, Prec. m*t. Acad. S~i. (Wash.), 71 (1974) 307-310. 20 Rose~mJ~_~, P,, MaCKF,', E. A., H~MAX', H. B., AYD DETTBARN, W. D., Choline acety[ase a n d cholinesterase activit5 in denervated electroplax, Biochim. biophys. Acre ~Amst.), 82 (1964) 266 275. 21 SHERIDAN, M. N., ] h e line structure of the electric organ of Torpedo marmorata. J. Cell Bh~l., 24 (1965) 129 141. 22 THeSt,VVF. S., Physiological effects of denervation of muscle, Attn. N . Y . Acad. Sci., 228 (1974) 89 104.