- Email: [email protected]
Neuraminidase: its effect on synaptic transmission
340
Brain Research, 80 (1974) 340 344 :(~ Elsevier Scientific Publishing Company, Amsterdam Printedin The Netherlands
Neuraminidase: its effect on synaptic transmission
L. TAUC AND D. H. HINZEN* Laboratoire de Neurobiologie Cellulaire, CNRS, 91190 Gijsur Yvette (France)
(Accepted July 30th, 1974)
Nervous tissue contains considerable amounts of complex materials, mainly gangliosides and sialoglycoproteins, having in common the presence of sialic acid among their constituents. Studies of the subcellular localization of sialic acid-containing substrates in the brain indicated that both gangliosidic and protein-bound sialic acid is specifically enriched in membranes of synaptosomes 1-zAl-lz,2°,21, thus suggesting an implication of these substrates in transmission processes. However, in spite of the rather extensive knowledge of the chemistry, biochemistry, and subcellular distribution of sialic acid-containing substrates no physiological approach has hitherto been tried successfully which could reveal a possible functional role of these substances in synaptic transmission. A degradation of sialic acid-containing substances can be effected by an enzyme, neuraminidase, which catalyzes the hydrolysis of gangliosidic and non-lipid-bound sialic acid. We were able to test a functional significance of siatic acid-containing substances in synaptic transmission by injecting neuraminidase into a presynaptic neuron and monitoring synaptic activity in a postsynaptic cell. We present results showing that neuraminidase introduced into the presynaptic cell progressively reduced, and finally completely abolished, postsynaptic responses without otherwise affecting the membrane properties of the pre- or postsynaptic neuron. All experiments were carried out in the buccal ganglion of Aplysia californica at room temperature. A pair of easily identifiable cholinergic interneurons and a postsynaptic cell being in inhibitory synaptic contact with both interneurons, and located close by in the same gangtion 4-6, were impaled with single- or double-barrelled glass microelectrodes filled with 2.5 M KCI. One interneuron was in addition impaled with a micropipette whose tip was filled with the neuraminidase solution. The second interneuron was used as a test. Purified neuraminidase (N-acetylneuraminate glycohydrolase, E.C. 3.2.1.18) from Clostridium perfringens was obtained from Sigma Chemical Co. (Type VI). The enzyme was successfully subjected to further chromatographic purification, and dissolved as a 5~o solution in sea water. Intraneuronal application was performed by using an air pressure system as previously described la. * Present address: Institute of Normal and Pathological Physiology, University of Cologne, G.F.R.
SHORT COMMUNICATIONS
341
S o m e experiments, carried o u t w i t h the n e u r a m i n i d a s e dissolved in t r i t i u m - l a b e l e d sea water, p e r m i t t e d us to calculate the injected v o l u m e as being a b o u t 1 ~ o f the cell s o m a volume. C o n t r o l e x p e r i m e n t s were p e r f o r m e d using, as injected substrates, ina c t i v a t e d n e u r a m i n i d a s e , proteases, a n d sea water alone. P o s t s y n a p t i c p o t e n t i a l s were p r o d u c e d b y direct electrical s t i m u l a t i o n o f one interneuron. A s h o r t t i m e before the m e a s u r e m e n t o f the p o s t s y n a p t i c p o t e n t i a l the m e m b r a n e p o t e n t i a l o f the p o s t s y n a p t i c cell was artificially h y p e r p o l a r i z e d to - - 8 0 mV. A t this m e m b r a n e p o t e n t i a l the posts y n a p t i c response was inverted to a d e p o l a r i z i n g p o t e n t i a l o f several m V a m p l i t u d e , which simplified the e x p e r i m e n t a l analysis. T h e g a n g l i o n was perfused with sea water t h r o u g h o u t the experiment. This p r o c e d u r e , o f injecting substances with pressure into a cell, does n o t by itself cause the cell m e m b r a n e a n y a p p r e c i a b l e d a m a g e . The a m p l i t u d e s o f the post-
BEFORE
/
A
2.5.ou~
B
¢
4 HOURS|
J
~ 44~40rnsef 5HOURS I ID
JIU/U 5.5 HOURS
C (,.-
neuraminidase
~
,~tllllLLlllll
E .200m~c
Fig. 1. Simultaneous intracellular recordings of spikes from a presynaptic neuron (upper traces of each set), and corresponding PSPs (lower traces) before and after intracellular injection of neuraminidase. The enzyme filled micropipette served also as an electrode for recording and stimulating of the presynaptic neuron through a 100 M f~ resistor. No compensating bridge circuitry was used. The small spike-like artifacts seen on the postsynaptic cell recordings, which are simultaneous with the spikes in the presynaptic neuron, are due to capaeitative coupling in the recording system. We did not try to minimize them in order to mark the time of firing. A: control recording before injection. In B, 2.5 h after injection the postsynaptic potential amplitudes started to decrease. In C and in D synaptic transmission is completely blocked. The spontaneous PSPs appearing in E after the end of the train indicate that the postsynaptic membrane remained sensitive to synaptic actions of other, noninjected interneurons. Vertical and horizontal calibrations in D and E respectively refer to D and E only. The schematic insert in each figure does not express actual distance or arrangement of pre- and postsynaptic cells.
342
SHORT COMMUNICATIONS
neurominidase
lo MIN
~
J
A 40 mVT 4mV140mseI
lest postsynaptk
postsynaptk
/ 2 HOURS
I II II B
) C
A ,os,~.1)~
.
postsynaptk
Fig. 2. Simultaneous intracellular recordings from an injected presynaptic interneuron (A and B, upper traces), a test interneuron (C, upper trace), and a cell synaptically connected to both interneurons (lower traces). A: control recording 10 rain after injection. B and C: 2 h after intracellular injection of neuraminidase (the recordings concerning the test at zero time, being essentially identical to those in C, are omitted). Note tbat the spike-like artifacts in the lower traces are intentionally increased. They do not indicate electrical coupling between the pre- and postsynaptic cells.
synaptic potentials started to decrease about 2-4 h after the intracellular application of the enzyme; the necessary time period being dependent upon the injected enzyme quantity. Then, 20-30 min later no synaptic activity at all could be induced by stimulation of the injected interneuron (Fig. 1). In contrast, no significant changes occurred in postsynaptic potential amplitudes due to stimulation of the noninjected or test intetneuron, nor in the resting potential of the postsynaptic cell (Fig. 2). Thus, no alteration in postsynaptic membrane properties could have induced the abolition of the synaptic activity. Only negligible changes were observed in the resting potential and the spike amplitude of the injected interneuron. The presynaptic endings were within a very short distance from the cell body permitting one to observe at the cell soma changes in spike conduction along the axon, and making it unlikely that suppressed synaptic activity was caused by a blockage of conduction. It may, therefore, be concluded that the complete abolition of synaptic transmission was due to the action of the neuraminidase at the nerve ending. Most of the delay in the obvious enzyme effect seems to be due to the axonal transport of the neuraminidase to the nerve ending. Since Sigma Chemical Co. indicated a small protease contamination (0.002 units/mg protein) in the original neuraminidase preparation, we ascertained whether this contamination could be responsible for the blocking effect on the synapse, However, even 100 times higher concentrations of protease were quite ineffective in this respect tg. Control experiments were also made with inactive neuraminidase and with sea water alone, to rule out the possibility of an action of low molecular weight
SHORT COMMUNICATIONS
343
contaminants. Synaptic activity was at no time influenced. It is very unlikely, therefore, that the neuraminidase effect is a nonspecific one. The effect of the neuraminidase on synaptic activity is susceptible to several interpretations. First, the enzyme may affect the mechanisms of transmitter synthesis and storage. However, prolonged inactivation of the presynaptic neuron, by hyperpolarization following the introduction of the neuraminidase, did not prevent complete suppression of synaptic activity within the same time period. Moreover, quite considerable storage of acetylcholine has been demonstrated in other Aplysia cholinergic synapseslL Also, sialic acid-containing substances do not appear to be important constituents of synaptic vesicles, being found only at a remarkably low content a,18,2°,21 or absent 2,11,14. A modification of the molecular organization of the vesicular membrane system affecting transmitter storage is, therefore, unlikely to occur. Second, the influx of calcium ions into the presynaptic terminal, necessary for the liberation of transmitterg, 1°, could be controlled by sialic acid-containing substances, and thus be inhibited by the action of the neuraminidase. We have considered this possibility in an indirect way: in order to obtain action potentials in Aplysia neurons the presence of both sodium and calcium is required 7,8A6. We found that action potentials could be produced in sodium-free sea water independently of whether the neuraminidase was present or not. In other words, as far as the somatic and axonic membranes are concerned, calcium entry is not prevented by neuraminidase. Any statement about the synaptic membrane would be purely speculative. Finally, we conclude from our results that the neuraminidase acts at the presynaptic terminal specifically by preventing release of transmitter. This hypothesis is consistent with the fact that sialic acid-containing compounds are definitely concentrated on the plasma membrane of the nerve endingl-3,11-13,2°,21. When applied externally the neuraminidase had no effect on synaptic activity. It may be that in this case the neuraminidase cannot produce an effect simply because it cannot penetrate to the synaptic region. Neuraminidase is specific to sialic acid-containing substances. However, both gangliosidic and non-lipid-bound sialic acids are susceptible to the action of this enzyme. Studies performed on the nervous tissue of different invertebrates revealed that gangliosides were lacking 17,18. Assuming this also to be true for Aplysia, it seems possible that the suppression of synaptic transmission is specifically related to the hydrolysis of sialoglycoproteins. The results demonstrated here seem to provide for the first time evidence of an important physiological role of sialic acid-containing substances in the nervous tissue. This work was supported by the Fondation pour la Recherche M6dicale Fran~aise, and by Grant No. 74.7.0205 from the DGRST (France) to L . T . D . H . H . was in receipt of a research fellowship from the Deutsche Forschungsgemeinschaft.
1 BRUNN~RAa~R,E. G., DEKIRMENJIAN,H., AND BROWN, B. D., The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain, Biochem. J., 103 (1967) 73-78.
344
SHORT COMMUNICATIONS
2 DEKIRMENJIAN,H., AND BRUNNGRABER,E. G., Distribution of protein-bound N-acetylneuraminic acid in subcellular particulate fractions prepared from rat whole brain, Biochim. biophy,~'. Acta (Amst.), 177 (1969) 1-10. 3 EICHBERG,J., WHITTAKER,V. P., AND DAWSON, R. M. C., The distribution of lipids in subcellular particles of guinea-pig brain, Biochem. J., 92 (1964) 91-100. 4 FIORE, L., ET MEUNIER, J.-M., Voles et relations neuronales dans le ganglion buccal de l'Aplysie, J. Physiol. (Paris), 67 (1973) 342A. 5 GARDNER, n., Bilateral symmetry and interneuronal organization in the buccal ganglion of Aplysia, Science, 173 (197l) 550-553. 6 GARDNER, n., ANn KANDEL, E. R., Diphasic postsynaptic potential: a chemical synapse capable of mediating conjoint excitation and inhibition, Science, 176 (1972) 675-678. 7 GEDULDIG, n., AND JUNGE, D., Sodium and calcium components of action potentials in the Aplysia giant neurone, J. Physiol. (Lond.), 199 (1968) 347-365. 8 JUNGE, D., Multi-ionic action potentials in molluscan giant neurones, Nature (Lond.), 215 (1967) 546-548. 9 KATZ, B., AND MILEDI, R., lonic requirements of synaptic transmitter release, Nature (Lond.), 215 (1967) 651. 10 KATZ, B., AND MILED1, R., Further study of the role of calcium in synaptic transmission, d. Physiol. (Lond.), 207 (1970) 789-801. 1l LAPETINA,E. G., SOTO, E. F,, AND DERoBERTIS, E., Gangliosides and acetylcholinesterase in isolated membranes of the rat brain cortex, Biochim. biophys. Acta (Amst.), 135 (1967) 33-43. 12 LAPETINA,E. G., SOTO, E. F., AND DEROBERTIS,E., Lipids and proteolipids in isolated subcellular membranes of rat brain cortex, J. Neurochem., 15 (1968) 437-445. 13 SEMINARIO,L. M., HREN, N., AND GOMEZ, C. J., Lipid distribution in subcellular fractions of the rat brain, J. Neuroehem., 11 (1964) 197-207. 14 SPENCE, M. W., AND WOLFE, L. S., Gangliosides in developing rat brain, Canad. J. Biochem., 45 (1967) 671-688. 15 STINNAKRE,J., Action de l'hdmicholinium sur une synapse centrale d'Aplysie, J, Physiol. (Paris), 62 (1970) 452-4-53. 16 STINNAKRE,J., AND TAUC, L., Calcium influx in active Aplysia neurones detected by injected aequorin, Nature New BioL, 242 (1973) 113-115. 17 SVENNERHOLM,L., Ganglioside metabolism. In M. FLORK1N AND E. H. STOTZ (Eds.), Comprehensive Biochemistry, Vol. 18, Elsevier, Amsterdam, 1970, pp. 201-227. 18 TAMAI, Y., ARAKI, S., KOMAI, Y., AND SATAKE, i . , Lipid distribution of the neuron, 4th Int. Meeting Int. Soc. Neurochem., Tokyo, 1973, p. 47. 19 TAU¢, L., HOFFMANN,A., TsuJI, S., HINZEN, n . H., AND FAILLE, L., Transmission abolished on a cholinergic synapse after injection of acetylcholinesterase into the presynaptic neurone, Nature (Lond.), 250 (1974) 496-498. 20 WHITTAKER,V. P., Some properties of synaptic membranes isolated from the central nervous system, Ann. N. Y. Acad. Sci., 137 (1966) 982-998. 21 WIEGANDT,H., The subcellular localization of gangliosides in the brain, J. Neurochem., 14 (1967) 671-674.
Brain Research, 80 (1974) 340 344 :(~ Elsevier Scientific Publishing Company, Amsterdam Printedin The Netherlands
Neuraminidase: its effect on synaptic transmission
L. TAUC AND D. H. HINZEN* Laboratoire de Neurobiologie Cellulaire, CNRS, 91190 Gijsur Yvette (France)
(Accepted July 30th, 1974)
Nervous tissue contains considerable amounts of complex materials, mainly gangliosides and sialoglycoproteins, having in common the presence of sialic acid among their constituents. Studies of the subcellular localization of sialic acid-containing substrates in the brain indicated that both gangliosidic and protein-bound sialic acid is specifically enriched in membranes of synaptosomes 1-zAl-lz,2°,21, thus suggesting an implication of these substrates in transmission processes. However, in spite of the rather extensive knowledge of the chemistry, biochemistry, and subcellular distribution of sialic acid-containing substrates no physiological approach has hitherto been tried successfully which could reveal a possible functional role of these substances in synaptic transmission. A degradation of sialic acid-containing substances can be effected by an enzyme, neuraminidase, which catalyzes the hydrolysis of gangliosidic and non-lipid-bound sialic acid. We were able to test a functional significance of siatic acid-containing substances in synaptic transmission by injecting neuraminidase into a presynaptic neuron and monitoring synaptic activity in a postsynaptic cell. We present results showing that neuraminidase introduced into the presynaptic cell progressively reduced, and finally completely abolished, postsynaptic responses without otherwise affecting the membrane properties of the pre- or postsynaptic neuron. All experiments were carried out in the buccal ganglion of Aplysia californica at room temperature. A pair of easily identifiable cholinergic interneurons and a postsynaptic cell being in inhibitory synaptic contact with both interneurons, and located close by in the same gangtion 4-6, were impaled with single- or double-barrelled glass microelectrodes filled with 2.5 M KCI. One interneuron was in addition impaled with a micropipette whose tip was filled with the neuraminidase solution. The second interneuron was used as a test. Purified neuraminidase (N-acetylneuraminate glycohydrolase, E.C. 3.2.1.18) from Clostridium perfringens was obtained from Sigma Chemical Co. (Type VI). The enzyme was successfully subjected to further chromatographic purification, and dissolved as a 5~o solution in sea water. Intraneuronal application was performed by using an air pressure system as previously described la. * Present address: Institute of Normal and Pathological Physiology, University of Cologne, G.F.R.
SHORT COMMUNICATIONS
341
S o m e experiments, carried o u t w i t h the n e u r a m i n i d a s e dissolved in t r i t i u m - l a b e l e d sea water, p e r m i t t e d us to calculate the injected v o l u m e as being a b o u t 1 ~ o f the cell s o m a volume. C o n t r o l e x p e r i m e n t s were p e r f o r m e d using, as injected substrates, ina c t i v a t e d n e u r a m i n i d a s e , proteases, a n d sea water alone. P o s t s y n a p t i c p o t e n t i a l s were p r o d u c e d b y direct electrical s t i m u l a t i o n o f one interneuron. A s h o r t t i m e before the m e a s u r e m e n t o f the p o s t s y n a p t i c p o t e n t i a l the m e m b r a n e p o t e n t i a l o f the p o s t s y n a p t i c cell was artificially h y p e r p o l a r i z e d to - - 8 0 mV. A t this m e m b r a n e p o t e n t i a l the posts y n a p t i c response was inverted to a d e p o l a r i z i n g p o t e n t i a l o f several m V a m p l i t u d e , which simplified the e x p e r i m e n t a l analysis. T h e g a n g l i o n was perfused with sea water t h r o u g h o u t the experiment. This p r o c e d u r e , o f injecting substances with pressure into a cell, does n o t by itself cause the cell m e m b r a n e a n y a p p r e c i a b l e d a m a g e . The a m p l i t u d e s o f the post-
BEFORE
/
A
2.5.ou~
B
¢
4 HOURS|
J
~ 44~40rnsef 5HOURS I ID
JIU/U 5.5 HOURS
C (,.-
neuraminidase
~
,~tllllLLlllll
E .200m~c
Fig. 1. Simultaneous intracellular recordings of spikes from a presynaptic neuron (upper traces of each set), and corresponding PSPs (lower traces) before and after intracellular injection of neuraminidase. The enzyme filled micropipette served also as an electrode for recording and stimulating of the presynaptic neuron through a 100 M f~ resistor. No compensating bridge circuitry was used. The small spike-like artifacts seen on the postsynaptic cell recordings, which are simultaneous with the spikes in the presynaptic neuron, are due to capaeitative coupling in the recording system. We did not try to minimize them in order to mark the time of firing. A: control recording before injection. In B, 2.5 h after injection the postsynaptic potential amplitudes started to decrease. In C and in D synaptic transmission is completely blocked. The spontaneous PSPs appearing in E after the end of the train indicate that the postsynaptic membrane remained sensitive to synaptic actions of other, noninjected interneurons. Vertical and horizontal calibrations in D and E respectively refer to D and E only. The schematic insert in each figure does not express actual distance or arrangement of pre- and postsynaptic cells.
342
SHORT COMMUNICATIONS
neurominidase
lo MIN
~
J
A 40 mVT 4mV140mseI
lest postsynaptk
postsynaptk
/ 2 HOURS
I II II B
) C
A ,os,~.1)~
.
postsynaptk
Fig. 2. Simultaneous intracellular recordings from an injected presynaptic interneuron (A and B, upper traces), a test interneuron (C, upper trace), and a cell synaptically connected to both interneurons (lower traces). A: control recording 10 rain after injection. B and C: 2 h after intracellular injection of neuraminidase (the recordings concerning the test at zero time, being essentially identical to those in C, are omitted). Note tbat the spike-like artifacts in the lower traces are intentionally increased. They do not indicate electrical coupling between the pre- and postsynaptic cells.
synaptic potentials started to decrease about 2-4 h after the intracellular application of the enzyme; the necessary time period being dependent upon the injected enzyme quantity. Then, 20-30 min later no synaptic activity at all could be induced by stimulation of the injected interneuron (Fig. 1). In contrast, no significant changes occurred in postsynaptic potential amplitudes due to stimulation of the noninjected or test intetneuron, nor in the resting potential of the postsynaptic cell (Fig. 2). Thus, no alteration in postsynaptic membrane properties could have induced the abolition of the synaptic activity. Only negligible changes were observed in the resting potential and the spike amplitude of the injected interneuron. The presynaptic endings were within a very short distance from the cell body permitting one to observe at the cell soma changes in spike conduction along the axon, and making it unlikely that suppressed synaptic activity was caused by a blockage of conduction. It may, therefore, be concluded that the complete abolition of synaptic transmission was due to the action of the neuraminidase at the nerve ending. Most of the delay in the obvious enzyme effect seems to be due to the axonal transport of the neuraminidase to the nerve ending. Since Sigma Chemical Co. indicated a small protease contamination (0.002 units/mg protein) in the original neuraminidase preparation, we ascertained whether this contamination could be responsible for the blocking effect on the synapse, However, even 100 times higher concentrations of protease were quite ineffective in this respect tg. Control experiments were also made with inactive neuraminidase and with sea water alone, to rule out the possibility of an action of low molecular weight
SHORT COMMUNICATIONS
343
contaminants. Synaptic activity was at no time influenced. It is very unlikely, therefore, that the neuraminidase effect is a nonspecific one. The effect of the neuraminidase on synaptic activity is susceptible to several interpretations. First, the enzyme may affect the mechanisms of transmitter synthesis and storage. However, prolonged inactivation of the presynaptic neuron, by hyperpolarization following the introduction of the neuraminidase, did not prevent complete suppression of synaptic activity within the same time period. Moreover, quite considerable storage of acetylcholine has been demonstrated in other Aplysia cholinergic synapseslL Also, sialic acid-containing substances do not appear to be important constituents of synaptic vesicles, being found only at a remarkably low content a,18,2°,21 or absent 2,11,14. A modification of the molecular organization of the vesicular membrane system affecting transmitter storage is, therefore, unlikely to occur. Second, the influx of calcium ions into the presynaptic terminal, necessary for the liberation of transmitterg, 1°, could be controlled by sialic acid-containing substances, and thus be inhibited by the action of the neuraminidase. We have considered this possibility in an indirect way: in order to obtain action potentials in Aplysia neurons the presence of both sodium and calcium is required 7,8A6. We found that action potentials could be produced in sodium-free sea water independently of whether the neuraminidase was present or not. In other words, as far as the somatic and axonic membranes are concerned, calcium entry is not prevented by neuraminidase. Any statement about the synaptic membrane would be purely speculative. Finally, we conclude from our results that the neuraminidase acts at the presynaptic terminal specifically by preventing release of transmitter. This hypothesis is consistent with the fact that sialic acid-containing compounds are definitely concentrated on the plasma membrane of the nerve endingl-3,11-13,2°,21. When applied externally the neuraminidase had no effect on synaptic activity. It may be that in this case the neuraminidase cannot produce an effect simply because it cannot penetrate to the synaptic region. Neuraminidase is specific to sialic acid-containing substances. However, both gangliosidic and non-lipid-bound sialic acids are susceptible to the action of this enzyme. Studies performed on the nervous tissue of different invertebrates revealed that gangliosides were lacking 17,18. Assuming this also to be true for Aplysia, it seems possible that the suppression of synaptic transmission is specifically related to the hydrolysis of sialoglycoproteins. The results demonstrated here seem to provide for the first time evidence of an important physiological role of sialic acid-containing substances in the nervous tissue. This work was supported by the Fondation pour la Recherche M6dicale Fran~aise, and by Grant No. 74.7.0205 from the DGRST (France) to L . T . D . H . H . was in receipt of a research fellowship from the Deutsche Forschungsgemeinschaft.
1 BRUNN~RAa~R,E. G., DEKIRMENJIAN,H., AND BROWN, B. D., The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain, Biochem. J., 103 (1967) 73-78.
344
SHORT COMMUNICATIONS
2 DEKIRMENJIAN,H., AND BRUNNGRABER,E. G., Distribution of protein-bound N-acetylneuraminic acid in subcellular particulate fractions prepared from rat whole brain, Biochim. biophy,~'. Acta (Amst.), 177 (1969) 1-10. 3 EICHBERG,J., WHITTAKER,V. P., AND DAWSON, R. M. C., The distribution of lipids in subcellular particles of guinea-pig brain, Biochem. J., 92 (1964) 91-100. 4 FIORE, L., ET MEUNIER, J.-M., Voles et relations neuronales dans le ganglion buccal de l'Aplysie, J. Physiol. (Paris), 67 (1973) 342A. 5 GARDNER, n., Bilateral symmetry and interneuronal organization in the buccal ganglion of Aplysia, Science, 173 (197l) 550-553. 6 GARDNER, n., ANn KANDEL, E. R., Diphasic postsynaptic potential: a chemical synapse capable of mediating conjoint excitation and inhibition, Science, 176 (1972) 675-678. 7 GEDULDIG, n., AND JUNGE, D., Sodium and calcium components of action potentials in the Aplysia giant neurone, J. Physiol. (Lond.), 199 (1968) 347-365. 8 JUNGE, D., Multi-ionic action potentials in molluscan giant neurones, Nature (Lond.), 215 (1967) 546-548. 9 KATZ, B., AND MILEDI, R., lonic requirements of synaptic transmitter release, Nature (Lond.), 215 (1967) 651. 10 KATZ, B., AND MILED1, R., Further study of the role of calcium in synaptic transmission, d. Physiol. (Lond.), 207 (1970) 789-801. 1l LAPETINA,E. G., SOTO, E. F,, AND DERoBERTIS, E., Gangliosides and acetylcholinesterase in isolated membranes of the rat brain cortex, Biochim. biophys. Acta (Amst.), 135 (1967) 33-43. 12 LAPETINA,E. G., SOTO, E. F., AND DEROBERTIS,E., Lipids and proteolipids in isolated subcellular membranes of rat brain cortex, J. Neurochem., 15 (1968) 437-445. 13 SEMINARIO,L. M., HREN, N., AND GOMEZ, C. J., Lipid distribution in subcellular fractions of the rat brain, J. Neuroehem., 11 (1964) 197-207. 14 SPENCE, M. W., AND WOLFE, L. S., Gangliosides in developing rat brain, Canad. J. Biochem., 45 (1967) 671-688. 15 STINNAKRE,J., Action de l'hdmicholinium sur une synapse centrale d'Aplysie, J, Physiol. (Paris), 62 (1970) 452-4-53. 16 STINNAKRE,J., AND TAUC, L., Calcium influx in active Aplysia neurones detected by injected aequorin, Nature New BioL, 242 (1973) 113-115. 17 SVENNERHOLM,L., Ganglioside metabolism. In M. FLORK1N AND E. H. STOTZ (Eds.), Comprehensive Biochemistry, Vol. 18, Elsevier, Amsterdam, 1970, pp. 201-227. 18 TAMAI, Y., ARAKI, S., KOMAI, Y., AND SATAKE, i . , Lipid distribution of the neuron, 4th Int. Meeting Int. Soc. Neurochem., Tokyo, 1973, p. 47. 19 TAU¢, L., HOFFMANN,A., TsuJI, S., HINZEN, n . H., AND FAILLE, L., Transmission abolished on a cholinergic synapse after injection of acetylcholinesterase into the presynaptic neurone, Nature (Lond.), 250 (1974) 496-498. 20 WHITTAKER,V. P., Some properties of synaptic membranes isolated from the central nervous system, Ann. N. Y. Acad. Sci., 137 (1966) 982-998. 21 WIEGANDT,H., The subcellular localization of gangliosides in the brain, J. Neurochem., 14 (1967) 671-674.