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Consequences of denervation on the distribution of the cholinergic (nicotinic) receptor sites fromElectrophorus electricus revealed by high resolution autoradiography
Brain Research, 62 (1973) 557-563
557
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
CONSEQUENCES OF DENERVATION ON THE DISTRIBUTION OF THE CHOL1NERGIC (NICOTINIC) RECEPTOR SITES FROM E L E C T R O P H O R U S E L E C T R I C U S REVEALED BY H I G H RESOLUTION A U T O R A D I O G R A P H Y
J. P. BOURGEOIS, J. L. POPOT, A. RYTER AND J. -P. CHANGEUX D~partement de Biologie Moldculaire, Institut Pasteur, Paris (France)
Snake venom a-toxins bind with a high selectivity and a low reversibility to cholinergic (nicotinic) receptor sites both in situ 8,16,25 and in vitro 8,11,21. They have, therefore, been widely used to characterize and localize these sites in fish electric organs ~,1°,11,19,2~, and vertebrate skeletal musclesla,22, 25. In a previous work 5, the distribution of cholinergic receptor sites in the electroplax from Electrophorus electricus was revealed by high resolution autoradiography with a tritiated a-toxin 18 from Naja nigricollis, and estimates of the number of toxin binding sites per square micron of cell surface in subsynaptic and extrasynaptic areas were proposed. Denervation of vertebrate skeletal muscle causes an increased sensitivity~,20 of extrasynaptic areas to cholinergic agonists accompanied by an increased number of toxin binding sites 9,11,13,22. Eel electric organ can be denervated easily by destruction of the caudal part of the spinal cord 27, and we have investigated whether a similar phenomenon occurs with this system. For up to 142 days after denervation, individual electroplax were dissected from the Sachs' organ 29. Some of them were incubated in [3H]a-toxin (10.5 Ci/mmole; 1.6/~g/ml in Ringer's solution) and prepared either for direct counting by liquid scintillation or for high resolution autoradiography following the previously described procedure 5. Others were used for electrophysiological and pharmacological studies. Some of the denervated organs were homogenized for in vitro titration of [3H]a-toxin binding sites 19. An electroplax is a giant syncytium which receives thousands of nerve terminals on its innervated surface. The nerve endings run in gutters on the electroplax surface and establish synaptic contacts from place to place. The stereology of the innervated membrane is rather complex 6. In extrasynaptic areas, microinvaginations of the plasma membrane increase the external surface by 10 ( ± 2)-fold while this increase is only I. 15 times in subsynaptic areas. On the whole, subsynaptic areas represent in Electrophorus electroplax about 2 ~ of the total surface of the innervated membrane. The first physiological change is observed 2 days after denervation: neurally evoked action potentials ('indirect' spikes) 1 and excitatory postsynaptic potentials disappear while action potentials elicited by 'direct' stimulation of the electroplax membrane 1 persist. Alterations of the ultrastructure of the nerve terminals accompany
558
J . P . BOURGEOIS ~'1 a/.
these physiological changes. Within 2 days most of the nerve endings degenerate. After 8 days the synaptic contacts represent only 0.1 ~o of the total surface of the innervated membrane instead of 2 ~ in the normal cell, and after a fortnight none of them is seen. No important morphological modification of the electroplax itself was noticed up to 142 days after denervation. An increase in membrane resistance, essentially due to decreased potassium permeability, is a common feature of denervated muscles 12,14,2°,24. The potassium conductance of the innervated membrane of the electroplax consists of an excitable component, with a non-linear I-V relation and a passive, linear leak conductance 2s. The excitable component is partially activated at the resting potential and inactivates during depolarizing currents z3. The two systems can be pharmacologically distinguished by bath application of barium. The leak conductance varies significantly among denervated electroplax but remains, on the average, close to that of innervated cells. In contrast, the excitable conductance diminishes reaching, after 10 days, about 40 ~ of the innervated controls. Consequently, the total resting conductance decreases slightly. During the same period, the resting potential does not change. Because the resting conductance is specific for potassium, it appears that the internal concentration of potassium near the membrane must vary by less than 1 - 2 ~ . More detailed electrophysiological results will be published later 6. It is noteworthy that in denervated electroplax, the increase in membrane resistance occurs despite the absence of denervation supersensitivity. Hence, the two modifications are not causally related. This conclusion can be tentatively extended to other species, where it has been found that the increases in sensitivity and resistance appear with different time courses2,3AL Axelsson and Thesleff4 and Miledi ~° found that the acetylcholine concentration in bath application needed to evoke a few mV depolarization or a twitch in denervated fibers from rat or frog muscles was from ten to several thousand times lower than that required by normal fibers. In contrast, bath application of decamethonium chloride, a cholinergic agonist, on isolated denervated electroplax 29 shows variations neither of the maximum depolarization nor of the doses necessary to induce an half-maximum response. Evolution of the total number of cholinergic receptor sites per electroplax after denervation was followed with the tritiated a-toxin by the method previously describedL Table I shows that this number does not change significantly up to 142 days after denervation. The same conclusion is reached by titration of the cholinergic receptor sites in vitro on crude homogenates of electric organ: 2, 3 and 4 weeks after denervation, the specific activity of membrane fragments separated by the method of Kasai and Changeux x5 is still close to 10-20 nmoles of [3H]a-toxin binding sites/g of protein 19. High resolution autoradiography brought further support to this conclusion. As already discussed (see also legend of Table I), if one makes several assumptions about the thickness of the thin sections, the exposure time, and the grain yield per tritium decay, densities of [all]a-toxin molecules bound/sq. # m of membrane surface can be estimated. In a control series of experiments, dissected cells were preincubated
0 (106) 8 (172) 15 (153) 28 (109) 52 (151) 142
Days after denervation
--
--
19.000 4. 7.200 (0.5 %) 21.500 4.4-6.000 (1.7 %) 20.000 + 7.000 (2 ,°4) 16.000 4. 4.000 (1.4%)
15.000 ! 6.000 (1.0 %) 20.000 4- 7.400 (0.4 %) ---
--
--
--
--
31.500 ± 10.000 (1.7%) (18.000) (0.1%) --
1.0 × 10 ~
240 4. 90
1.1 × 1011
1.I × 1011
220 4. 120
240 4- 60
1.0 × 1011
2.6 × 1011
124 4. 50
300 ± 200
Extrasynaptic
ct]
2.0 × 1011
2.0 × 1011
1.2 × 1011
2.3 × 1011
2.6 × 1011
m ~d
X 0 X o ©
.< ,..]
Z7 t~ Z
(2 4. 0.5) × 1011
Measured directly by liquid scintillation
Calculated from autoradiography
Sub-Schwann.
Subsynaptic
Free clusters
Total labeled sites per cell
Number o f [3H]a-toxin molecules bound/sq. #m o f plasma membrane surface
For the high resolution autoradiography silver colored sections were used corresponding to about 0.05/~m in thickness and controlled with an interferometer, from the thickness of the thin sections, the exposure time at 20 °C (numbers of days in parentheses), and the silver grain densities measured on micrographs, a number of grains/min/sq./~m on cell surface was estimated. The yield of grains to tritium disint./min was assumed to be 20 %7 in order to calculate the number of tritiated a-toxin labeled sites/sq. #m of membrane surface, taking into account the complex stereology of this surface. The percentage in parentheses indicate the subsynaptic, sub-Schwannian and free cluster surfaces compared to the total real surface of the innervated face of the electroplax.
TABLE I
,q ©
t~
h
u~
EFFECT OF DENERVATIONON CHOLINERGICRECEPTOR SITES
561
in a solution either of unlabeled a-toxin (5/~g/ml) or of an irreversible antagonist (10-4M TDF), prior to normal incubation with [SH]a-toxin. The number of toxin molecules bound after treatment by T D F is in the same range as that found after preincubation with unlabeled a-toxin and assumed to represent non-specific labeling. It consists of 8 ~ of the total amount of toxin molecules bound under the synapses, 20-30 ~ of those bound in the extrasynaptic areas, and 100 ~ of those associated with the non-innervated surface. This non-specific binding does not change after denervation. In the normal electroplax, high grain densities are observed only under the nerve endings (Fig. 1A). No free clusters of toxin binding sites are seen between the synapses. 8 days after denervation, as already mentioned, only 0.1 ~ of the nerve endings remain. However, empty synaptic gutters can still be identified by their shape although nerve terminals are absent (Fig. 1B). In these gutters the density of grains, and therefore that of toxin binding sites, is very close to that observed in normally innervated subsynaptic membranes. A fortnight after denervation such 'free' clusters are still present (Fig. 1C). The analysis of serial sections confirmed that the observed clusters are freely exposed to the cell environment. No residual nerve processes were ever seen in close contact with or in the vicinity of these clusters 8 and 15 days after denervation. One month after denervation Schwann cell processes covered all the clusters observed (Fig. 1C). Because of their dimensions, density and distribution, these grain clusters were interpreted as representing the remnants of subsynaptic structure persisting at least 142 days after denervation. There is good evidence that, in the electroplax, toxin binding sites are the cholinergic receptor sites. The persistence of cholinergic receptor grouping for days and weeks without any tendency for lateral diffusion suggests that, at least, at the level of the subsynaptic membrane the fluidity of the cytoplasmic membrane is not as high as that expected from experiments with spin-labeled lipids 17. On the other hand, special factors might prevent lateral diffusion of the cholinergic receptor protein: the tendency to form lattice structures or the presence of 'cleft substances' which would cross-link neighboring receptor molecules. In this regard an electrondense layer present on the surface of the subsynaptic membrane in the normal electroplax seems to persist several weeks after denervation in the areas of receptor clusters (Fig. 1C).
Fig. 1. Electron microscope autoradiography of the electroplax from Electrophorus electricus labeled by laH]a-toxin from Naja nigricollis. Each silver grain represents one tritium source, that is to say in our case one toxin molecule bound to the cell surface. A" the normal electroplax receives several thousands of nerve endings (NE) making synaptic contacts (arrow) with the cell surface. The silver grains of the subsynaptic areas are lccated near and along the synaptic cleft forming silver grain clusters. B: 8 days after denervation the nerve ending is absent whereas the synaptic gutter (arrow) remains intact. The 'free' silver grains are yet clustered and their distribution and density are quite similar to those observed in the non-denervated synapses. C: 15 days after denervation the 'free' silver grain clusters are still observable. The synaptic gutter shape is intact (arrow). D: 15 days after denervation, Schwann cell (S) processes begin to cover the synaptic gutters with associated silver grain clusters (arrows). 28 days and later after denervation all the subsynaptic areas are covered with such Schwann cell processes. Scale marker, ! btm.
562
J . P . BOURGEOIS el a[.
In conclusion: with the electroplax, in c o n t r a s t to what is seen with skeletal muscles o f a m p h i b i a n s ")° and mammals4,13, the ratio o f extrasynaptic to subsynaptic a - t o x i n binding sites does not change significantly until 52 days after denervation. The n o n - a p p e a r a n c e o f new r e c e p t o r sites is further s u p p o r t e d by electrophysiological d a t a a n d in vitro titrations on crude h o m o g e n a t e s . One o f the most striking results o f d e n e r v a t i o n in this system is the persistence o f p o s t s y n a p t i c structures, synaptic gutters and cholinergic r e c e p t o r site clusters, long after the d i s a p p e a r a n c e o f nerve terminals. The absence o f lateral diffusion o f the r e c e p t o r protein observed in these c o n d i t i o n s suggests that the p o s t s y n a p t i c m e m b r a n e possesses a p a r t i c u l a r l y stable a n d rigid structure. Dr. P. B o q u e t deserves special t h a n k s for supplying the a - t o x i n o f N a j a nigricollis, and Drs. A. Menez, J. L. M o r g a t and P. F r o m a g e o t for its tritiation. This w o r k was s u p p o r t e d by funds from the Centre N a t i o n a l de la Recherche Scientifique, the D616gation G6n6rale /t la Recherche Scientifique et Technique ( N O . 71.7.3082 a n d 71.7.3040), the Coll~ge de France, the C o m m i s s a r i a t h l'Energie A t o m i q u e a n d N a t i o n a l Institutes o f Health.
1 ALBE-FESSARD, D., CHAGAS, C., ET MARTIN-FERREIRA, H., Sur l'existence d'un syst~me directement excitable dans l'organe 61ectrique de l'Electrophorus electricus, C.R. Acad. Sci. (Paris), 232 (1951) 1015-1017. 2 ALBUQUERQUE,E. X., AND MC1SAAC, R. J., Fast and slow mammalian muscle after denervation, Exp. Neurol., 26 (1970) 183-202. 3 ALBUQUERQUE,E. X., SCHUH, F. T., AND KAUFFMAN, l7. C., Early membrane depolarization of the
fast mammalian muscle after denervation, Pfliigers Arch. ges. Physiol., 328 (1971) 36-50. 4 AXELSSON,J., AND THESLEEF, S., A study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. (Lond.), 149 (1959) 178-193. 5 BOURGEOIS, J. P., RYTER, A., MENEZ, A., FROMAGEOT, P., BOQUET, P., AND CHANGEUX, .l.P.,
Localization of the cholinergic receptor protein in Electrophorus electroplax by high resolution autoradiography, FEBS Letters, 25 (1972) 127-133. 6 BOURGEOIS,J. P., POPOT, J. L., RYTER, A., AND CHANGEUX, J. P., In preparation. 7 CARO,L. G., Considerations on high resolution autoradiography, J. roy. Microsc. Soc., 83 (1964) 127-133. 8 CHANGEUX, J. P., KASAI, M., AND LEE, C. Y., Use of a snake venom toxin to characterize the cholinergic receptor protein, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 1241-1247. 9 CHIU, T. H., DOLLY,J. O., AND BARNARD,E. A., Solubilization from skeletal muscle of two components that specifically bind a-bungarotoxin, Biochem. biophys. Res. Commun., 51 (1973) 205213. I0 FULPIUS,B., CHA, S., KLETT, R., AND REICH, E., Properties of the nicotinic acetylcholine receptor macromolecule of Electrophorus electricus, FEBS Letters, 24 (1972) 323-326. 11 HALL, Z. W., Release of neurotransmitters and their interaction with receptors, Ann. Rev. Biochem., 41 (1972) 925-952. 12 HARRIS, E. J., AND NICHOLLS, J. G., The effect of denervation on the rate of entry of potassium into frog muscle, J. Physiol. (Lond.), 131 (1956) 473-476. 13 HARTZELL, H. C., AND FAMBROUGH, D. M., Acetylcholine receptors: distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity, J. gen. Physiol., 60 (1972) 248-262. 14 KLAUS,W., LCLLMAN,H., UND MUSCHOLL,E., Der Kalium Flux des normale und denervierten Rattenzwerchfells, Pfliigers Arch. ges. Physiol., 271 (1960) 761-775. 15 KASAI,M., AND CHANGEUX,J. P., In vitro excitation of purified membrane fragments by cholinergic agonists, J. Membrane Biol., 6 (1971) 1-88.
EFFECT OF DENERVATION ON CHOLINERGIC RECEPTOR SITES
563
16 LEE, C. Y., CHANG, C. C., AND CHEN, Y. M., Reversibility of neuromuscular blockade by neurotoxins from elapead and sea snake venoms, J. Formosan Med. Ass., 71 (1972) 344-349. 17 McCONNEL, M., DEVAUX, P., AND SCANDELLA,C., Lateral diffusion and phase separations in biological membranes In Membrane Research, Academic Press, New York, 1972, pp. 27-37. 18 MENEZ, A., MORGAT,J. L., FROMAGEOT,P., RONSERAY,A. M., BOQUET,P.: AND CHANGEUX,J. P., Tritium labelling of the a-neurotoxin of Naja nigricollis, FEBS Letters, 17 (1971) 333-335. 19 MEUNIER,J. C., OLSEN, R. W., MENEZ, A., FROMAGEOT,P., BOQUET, P., AND CHANGEUX,J. P., Some physical properties of the cholinergic receptor protein from Electrophorus electricus revealed by a tritiated a-toxin from Naja nigricollis venom, Biochemistry, 11 (1972) 1200-1210. 20 MILEDI, R., The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation, J. Physiol. (Lond.), 151 (1960) 1-23. 21 MILEDI, R., MOLINOEF, P., AND POTTER, L. T., Isolation of the cholinergic receptor protein of Torpedo electric tissue, Nature (Lond.), 229 (1971) 554-557. 22 MILEDI, R., AND POTTER, L. T., Acetylcholine receptors in muscle fibers, Nature (Lond.), 233 (1971) 599-603. 23 NAKAMURA,Y., NAKAJIMA,S., AND GRUNDEEST,H., Analysis of spike electrogenesis and depolarizing K inactivation in electroplax of Electrophorus electricus L., J. gen. Physiol., 49 (1965) 321349. 24 NICHOLLS,J. G., The electrical properties of denervated skeletal muscle, J. Physiol. (Lond.), 131 (1956) 1-12. 25 PORTER,C. W., CHIU, T. H., WIECKOWSKI,J., AND BARNARD,E. A., Types and locations of cholinergic receptor like molecules in muscle fibres, Nature New Biol., 241 (1973) 3-7. 26 RAFTERY,M. A., SCHMIDT, J., CLARK, O. G., AND WOLCOTT, R. G., Demonstration of a specific a-bungarotoxin binding component in Electrophorus electricus electroplax membranes, Biochem. biophys. Res. Commun., 45 (1971) 1622-1629. 27 ROSENBERG,P., McKEY, E. A., HIGMAN,H. B., AND DETTBARN,W. O., Choline acetylase and cholinesterase activity in denervated electroplax, Biochim. biophys. Acta (Amst.), 82 (1964) 266-275. 28 RUIZ-MANRESA,F., RUARTE, A. C., SCHWARTZ, T. L., AND GRUNDFEST,H., Potassium inactivation and impedance changes during spike electrogenesis in eel electroplax, J. gen. Physiol., 55 (1970) 33-47. 29 SCHOEFENIELS,E., AND NACHMANSOHN,D., An isolated single electroplax preparation. I. New data on the effect of acetylcholine and related compounds, Biochim. biophys. Acta (Amst.), 26 (1957) 1-15.
557
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
CONSEQUENCES OF DENERVATION ON THE DISTRIBUTION OF THE CHOL1NERGIC (NICOTINIC) RECEPTOR SITES FROM E L E C T R O P H O R U S E L E C T R I C U S REVEALED BY H I G H RESOLUTION A U T O R A D I O G R A P H Y
J. P. BOURGEOIS, J. L. POPOT, A. RYTER AND J. -P. CHANGEUX D~partement de Biologie Moldculaire, Institut Pasteur, Paris (France)
Snake venom a-toxins bind with a high selectivity and a low reversibility to cholinergic (nicotinic) receptor sites both in situ 8,16,25 and in vitro 8,11,21. They have, therefore, been widely used to characterize and localize these sites in fish electric organs ~,1°,11,19,2~, and vertebrate skeletal musclesla,22, 25. In a previous work 5, the distribution of cholinergic receptor sites in the electroplax from Electrophorus electricus was revealed by high resolution autoradiography with a tritiated a-toxin 18 from Naja nigricollis, and estimates of the number of toxin binding sites per square micron of cell surface in subsynaptic and extrasynaptic areas were proposed. Denervation of vertebrate skeletal muscle causes an increased sensitivity~,20 of extrasynaptic areas to cholinergic agonists accompanied by an increased number of toxin binding sites 9,11,13,22. Eel electric organ can be denervated easily by destruction of the caudal part of the spinal cord 27, and we have investigated whether a similar phenomenon occurs with this system. For up to 142 days after denervation, individual electroplax were dissected from the Sachs' organ 29. Some of them were incubated in [3H]a-toxin (10.5 Ci/mmole; 1.6/~g/ml in Ringer's solution) and prepared either for direct counting by liquid scintillation or for high resolution autoradiography following the previously described procedure 5. Others were used for electrophysiological and pharmacological studies. Some of the denervated organs were homogenized for in vitro titration of [3H]a-toxin binding sites 19. An electroplax is a giant syncytium which receives thousands of nerve terminals on its innervated surface. The nerve endings run in gutters on the electroplax surface and establish synaptic contacts from place to place. The stereology of the innervated membrane is rather complex 6. In extrasynaptic areas, microinvaginations of the plasma membrane increase the external surface by 10 ( ± 2)-fold while this increase is only I. 15 times in subsynaptic areas. On the whole, subsynaptic areas represent in Electrophorus electroplax about 2 ~ of the total surface of the innervated membrane. The first physiological change is observed 2 days after denervation: neurally evoked action potentials ('indirect' spikes) 1 and excitatory postsynaptic potentials disappear while action potentials elicited by 'direct' stimulation of the electroplax membrane 1 persist. Alterations of the ultrastructure of the nerve terminals accompany
558
J . P . BOURGEOIS ~'1 a/.
these physiological changes. Within 2 days most of the nerve endings degenerate. After 8 days the synaptic contacts represent only 0.1 ~o of the total surface of the innervated membrane instead of 2 ~ in the normal cell, and after a fortnight none of them is seen. No important morphological modification of the electroplax itself was noticed up to 142 days after denervation. An increase in membrane resistance, essentially due to decreased potassium permeability, is a common feature of denervated muscles 12,14,2°,24. The potassium conductance of the innervated membrane of the electroplax consists of an excitable component, with a non-linear I-V relation and a passive, linear leak conductance 2s. The excitable component is partially activated at the resting potential and inactivates during depolarizing currents z3. The two systems can be pharmacologically distinguished by bath application of barium. The leak conductance varies significantly among denervated electroplax but remains, on the average, close to that of innervated cells. In contrast, the excitable conductance diminishes reaching, after 10 days, about 40 ~ of the innervated controls. Consequently, the total resting conductance decreases slightly. During the same period, the resting potential does not change. Because the resting conductance is specific for potassium, it appears that the internal concentration of potassium near the membrane must vary by less than 1 - 2 ~ . More detailed electrophysiological results will be published later 6. It is noteworthy that in denervated electroplax, the increase in membrane resistance occurs despite the absence of denervation supersensitivity. Hence, the two modifications are not causally related. This conclusion can be tentatively extended to other species, where it has been found that the increases in sensitivity and resistance appear with different time courses2,3AL Axelsson and Thesleff4 and Miledi ~° found that the acetylcholine concentration in bath application needed to evoke a few mV depolarization or a twitch in denervated fibers from rat or frog muscles was from ten to several thousand times lower than that required by normal fibers. In contrast, bath application of decamethonium chloride, a cholinergic agonist, on isolated denervated electroplax 29 shows variations neither of the maximum depolarization nor of the doses necessary to induce an half-maximum response. Evolution of the total number of cholinergic receptor sites per electroplax after denervation was followed with the tritiated a-toxin by the method previously describedL Table I shows that this number does not change significantly up to 142 days after denervation. The same conclusion is reached by titration of the cholinergic receptor sites in vitro on crude homogenates of electric organ: 2, 3 and 4 weeks after denervation, the specific activity of membrane fragments separated by the method of Kasai and Changeux x5 is still close to 10-20 nmoles of [3H]a-toxin binding sites/g of protein 19. High resolution autoradiography brought further support to this conclusion. As already discussed (see also legend of Table I), if one makes several assumptions about the thickness of the thin sections, the exposure time, and the grain yield per tritium decay, densities of [all]a-toxin molecules bound/sq. # m of membrane surface can be estimated. In a control series of experiments, dissected cells were preincubated
0 (106) 8 (172) 15 (153) 28 (109) 52 (151) 142
Days after denervation
--
--
19.000 4. 7.200 (0.5 %) 21.500 4.4-6.000 (1.7 %) 20.000 + 7.000 (2 ,°4) 16.000 4. 4.000 (1.4%)
15.000 ! 6.000 (1.0 %) 20.000 4- 7.400 (0.4 %) ---
--
--
--
--
31.500 ± 10.000 (1.7%) (18.000) (0.1%) --
1.0 × 10 ~
240 4. 90
1.1 × 1011
1.I × 1011
220 4. 120
240 4- 60
1.0 × 1011
2.6 × 1011
124 4. 50
300 ± 200
Extrasynaptic
ct]
2.0 × 1011
2.0 × 1011
1.2 × 1011
2.3 × 1011
2.6 × 1011
m ~d
X 0 X o ©
.< ,..]
Z7 t~ Z
(2 4. 0.5) × 1011
Measured directly by liquid scintillation
Calculated from autoradiography
Sub-Schwann.
Subsynaptic
Free clusters
Total labeled sites per cell
Number o f [3H]a-toxin molecules bound/sq. #m o f plasma membrane surface
For the high resolution autoradiography silver colored sections were used corresponding to about 0.05/~m in thickness and controlled with an interferometer, from the thickness of the thin sections, the exposure time at 20 °C (numbers of days in parentheses), and the silver grain densities measured on micrographs, a number of grains/min/sq./~m on cell surface was estimated. The yield of grains to tritium disint./min was assumed to be 20 %7 in order to calculate the number of tritiated a-toxin labeled sites/sq. #m of membrane surface, taking into account the complex stereology of this surface. The percentage in parentheses indicate the subsynaptic, sub-Schwannian and free cluster surfaces compared to the total real surface of the innervated face of the electroplax.
TABLE I
,q ©
t~
h
u~
EFFECT OF DENERVATIONON CHOLINERGICRECEPTOR SITES
561
in a solution either of unlabeled a-toxin (5/~g/ml) or of an irreversible antagonist (10-4M TDF), prior to normal incubation with [SH]a-toxin. The number of toxin molecules bound after treatment by T D F is in the same range as that found after preincubation with unlabeled a-toxin and assumed to represent non-specific labeling. It consists of 8 ~ of the total amount of toxin molecules bound under the synapses, 20-30 ~ of those bound in the extrasynaptic areas, and 100 ~ of those associated with the non-innervated surface. This non-specific binding does not change after denervation. In the normal electroplax, high grain densities are observed only under the nerve endings (Fig. 1A). No free clusters of toxin binding sites are seen between the synapses. 8 days after denervation, as already mentioned, only 0.1 ~ of the nerve endings remain. However, empty synaptic gutters can still be identified by their shape although nerve terminals are absent (Fig. 1B). In these gutters the density of grains, and therefore that of toxin binding sites, is very close to that observed in normally innervated subsynaptic membranes. A fortnight after denervation such 'free' clusters are still present (Fig. 1C). The analysis of serial sections confirmed that the observed clusters are freely exposed to the cell environment. No residual nerve processes were ever seen in close contact with or in the vicinity of these clusters 8 and 15 days after denervation. One month after denervation Schwann cell processes covered all the clusters observed (Fig. 1C). Because of their dimensions, density and distribution, these grain clusters were interpreted as representing the remnants of subsynaptic structure persisting at least 142 days after denervation. There is good evidence that, in the electroplax, toxin binding sites are the cholinergic receptor sites. The persistence of cholinergic receptor grouping for days and weeks without any tendency for lateral diffusion suggests that, at least, at the level of the subsynaptic membrane the fluidity of the cytoplasmic membrane is not as high as that expected from experiments with spin-labeled lipids 17. On the other hand, special factors might prevent lateral diffusion of the cholinergic receptor protein: the tendency to form lattice structures or the presence of 'cleft substances' which would cross-link neighboring receptor molecules. In this regard an electrondense layer present on the surface of the subsynaptic membrane in the normal electroplax seems to persist several weeks after denervation in the areas of receptor clusters (Fig. 1C).
Fig. 1. Electron microscope autoradiography of the electroplax from Electrophorus electricus labeled by laH]a-toxin from Naja nigricollis. Each silver grain represents one tritium source, that is to say in our case one toxin molecule bound to the cell surface. A" the normal electroplax receives several thousands of nerve endings (NE) making synaptic contacts (arrow) with the cell surface. The silver grains of the subsynaptic areas are lccated near and along the synaptic cleft forming silver grain clusters. B: 8 days after denervation the nerve ending is absent whereas the synaptic gutter (arrow) remains intact. The 'free' silver grains are yet clustered and their distribution and density are quite similar to those observed in the non-denervated synapses. C: 15 days after denervation the 'free' silver grain clusters are still observable. The synaptic gutter shape is intact (arrow). D: 15 days after denervation, Schwann cell (S) processes begin to cover the synaptic gutters with associated silver grain clusters (arrows). 28 days and later after denervation all the subsynaptic areas are covered with such Schwann cell processes. Scale marker, ! btm.
562
J . P . BOURGEOIS el a[.
In conclusion: with the electroplax, in c o n t r a s t to what is seen with skeletal muscles o f a m p h i b i a n s ")° and mammals4,13, the ratio o f extrasynaptic to subsynaptic a - t o x i n binding sites does not change significantly until 52 days after denervation. The n o n - a p p e a r a n c e o f new r e c e p t o r sites is further s u p p o r t e d by electrophysiological d a t a a n d in vitro titrations on crude h o m o g e n a t e s . One o f the most striking results o f d e n e r v a t i o n in this system is the persistence o f p o s t s y n a p t i c structures, synaptic gutters and cholinergic r e c e p t o r site clusters, long after the d i s a p p e a r a n c e o f nerve terminals. The absence o f lateral diffusion o f the r e c e p t o r protein observed in these c o n d i t i o n s suggests that the p o s t s y n a p t i c m e m b r a n e possesses a p a r t i c u l a r l y stable a n d rigid structure. Dr. P. B o q u e t deserves special t h a n k s for supplying the a - t o x i n o f N a j a nigricollis, and Drs. A. Menez, J. L. M o r g a t and P. F r o m a g e o t for its tritiation. This w o r k was s u p p o r t e d by funds from the Centre N a t i o n a l de la Recherche Scientifique, the D616gation G6n6rale /t la Recherche Scientifique et Technique ( N O . 71.7.3082 a n d 71.7.3040), the Coll~ge de France, the C o m m i s s a r i a t h l'Energie A t o m i q u e a n d N a t i o n a l Institutes o f Health.
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