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Heterogeneity of synaptic vesicles in the squid giant fibre system
BRAIN RESEARCH
573
HETEROGENEITY OF SYNAPTIC VESICLES IN THE SQUID GIANT FIBRE SYSTEM
D I E T E R FROESCH AND R A I N E R M A R T I N
Neurobiology Section, Stazione Zoologica di Napoli, Naples (Italy) (Accepted February 25th, 1972)
INTRODUCTION
The squid giant fibre system with its 3 sets of identified neurones4,15 offers the opportunity to compare synaptic fine structure in 4 successive contacts from the afferent input in the brain through two giant synapses in the palliovisceral lobe and the stellate ganglion. The most peripheral synapse in this chain, the neuromuscular junction between the third order giant axon and the mantle muscles, has not yet been identified. In the present study we examine the average vesicle size in populations of agranular vesicles in 5 synaptic contacts of the giant fibre system and in the fin musculature. The reactivity with zinc iodide-osmium (ZIO) is used as a further criterion to distinguish the synaptic vesicles of the 5 contacts. The results show that there are 4 significantly different sized classes of synaptic vesicles, that 3 of the vesicle populations react with ZIO, and that two do not. If these variations in vesicle size and reactivity reflect chemical differences between the synapses, one may postulate that in the giant fibre system there are 4 different mechanisms of synaptic transmission. At present the chemical nature of the transmitter substance has not been established in any of the contacts. MATERIAL AND METHODS
Specimens ofLoligo vulgaris were fixed by perfusion with 1% OsO4 in sea-water with 0.1 M cacodylate buffer (pH 7.4) or in a cephalopod Ringer solution9. Synapses (d) and (e) (see Results) were fixed by immersion of the stellate ganglion and fin muscles into 1.5~o OsOa dissolved in sea-water with 0.1 M cacodylate buffer. The vesicles of synapse (d) were measured from 3 animals of different mantle lengths (5, 12, 20 cm) that were fixed by perfusion or immersion. The vesicle diameters of this material are regularly distributed, as plotted in Fig. 2D. ZIO was applied as described in ref. 10~The tissues were dehydrated in ethanol and propylene oxide, and embedded in Durcupan. The sections were stained with lead citrate. The magnification of the electron microscope (Philips EM 200) was calibrated with a carbon replica of a diffrac-
Brain Research, 43 (1972) 573-579
574
D. F R O E S C t l A N D R. M A R T I N
tion grating (Ladd Research Inc.). The diameter o f the vesicles including the membrane was measured on electron micrographs × 88,000. In oval vesicles the smallest and the largest diameter were measured and the mean o f the two diameters was used for the statistics. The significance o f the size differences in the 5 vesicle populations was demonstrated by a t-test. RESULTS Fig. 1 represents schematically the connexions o f the giant fibre system o f the squid. The 5 synapses studied were: (a) afferent synaptic b o u t o n s on the cell b o d y o f the first order giant cell in the magnocellular lobe o f the brain4; (b) synaptic b o u t o n s on the distal first order giant axon in the palliovisceral lobe next to its contact with the second order giant axon4; (c) the axo-axonal contact o f the first order with the second order giant axon in the palliovisceral lobe4; (d) the axo-axonal contact o f the second order with the third order giant axon in the stellate ganglion11; and (e) neuromuscular junctions on muscle fibres o f the fin.
.U
a ,L
ax
%o
~x
a × II
~x
II
oOc~7~D
,~x III
e
Fig. 1. Left side, a schematic representation of the synaptic contacts in the giant fibre system; a, b, c, d and e are the synapses examined in this study (see text). I, II, III, first, second and third order giant neurones. Right side, a schematic drawing of the structural polarity of the 5 contacts (see text). I, first order giant cell; ax I, ax II, ax III, first, second and third order giant axons. Brain Research, 43 (1972) 573-579
575
SYNAPTIC VESICLES IN SQUID GIANT AXONS
o
lOO-i
m i
o 100
~0
0-
nn
-
in--m--
~
-I
d
27
J4
41
4~
,~S
62
~9
16
B~$
90
97
102 qr~ diameter
Fig. 2. Size distribution (nm) of vesicle diameter in the 5 synapses of Fig. 1. In b the vesicles inside the afferent bouton are represented. Contacts (a), (b), (d) and (e) have the structural characteristics of chemical synapses, i.e. a large synaptic cleft and masses of synaptic vesicles near the presynaptic membrane. Contact (c) shows the structural characteristics of chemical as well as of electrical synapses, the latter characterized by lack of synaptic vesicles and a gap junction of the membranes with a cleft smaller than 5 nm 4. While contacts (a), (c), (d) and (e) show accumulations of vesicles on one side only, contact (b) is characterized by masses of synaptic vesicles of different size on both sides of the synaptic membranes4. We do not have evidence that the neuromuscular contacts (e) are the terminals of giant axons. The structural polarity of the 5 contacts and the width of the synaptic clefts are schematically drawn in Fig. 1. The size distributions of the vesicles are shown in Fig. 2. The variation of vesicle size is highest in contact (c), but similar in contacts (a), (b), (d) and (e). The measured diameters of a larger number of synaptic vesicles in the 5 contacts are reported in Table I. The values are not significantly different between synapses (a) and (e), but between all the other synapses there are significant differences. Fig. 3 shows electron micrographs of vesicle populations of the 5 contacts not treated (left column) or treated (right column) with ZIO. The large majority of vesicles of contacts (c) and (d) do not react with ZIO, although a few vesicular profiles with reaction product are present. A control for the successful reaction consists in contact Brain Research, 43 (1972) 573-579
576
D. FROESCH ANI) R. MARTIN
Fig. 3. Electron m i c r o g r a p h s ( 7, 100,000) o f 5 vesicle populations in the synapses of Fig. 1. C o n t a c t b also c o n t a i n s vesicles on the side o f the first order axon, as s h o w n in c.
Brain Research, 43 (1972) 573-579
577
S Y N A P T I C VESICLES I N S Q U I D G I A N T A X O N S
TABLE
I
STATISTICS OF THE VESICLE DIAMETER OF THE 5 SYNAPSES
Synaptic contact
Number of vesicles
vesicle
Standard deviation:
Standard error:
measured: n
diameter:
s (nm)
s~ (nm)
Mean o f
(rim) a
369
42.5
5.1
0.014
b
427
51.2
6.6
0.015
c
610
69.1
12.8
0.021
d
601
54.4
5.2
0.008
e
499
43.5
6.9
0.014
Significance of the differences in means between two populations: t-test
a:b t a:c t a:d t a:e. t b:c t b:d t b:e t
= = = = = = = c:d t = C:C t = d :e, t =
21.5 P < 0.005 58.3 P < 0 . 0 0 5 73.4 P < 0.005 2.33 P = 0.01 39.7 P < 0.005 8.6 P < 0.005 17.6P < 0.005 38.5 P < 0.005 57.7 P < 0.005 30.3 P < 0 . 0 0 5
(b) in which a reactive and an unreactive population lie on either side of the same synaptic membrane pair 10. The population of large vesicles in contact (c) is identical with the population of vesicles on the side of the first order giant axon in contact (b). DISCUSSION
By comparison of the average size of 5 populations of agranular electrontransparent synaptic vesicles in 5 different synaptic contacts, 4 of which belong to identified neurones of the giant fibre system, we can distinguish 4 different size classes. In two populations the difference in means is probably not significant: the vesicles of afferent synaptic boutons on the first order giant neurone in the brain (a) and the vesicles of neuromuscular junctions in the fin (e). Both these populations react with ZIO. The 3 others, all with larger vesicles, belong to significantly different size classes. They are further distinguished by their reactivity with ZIO. The vesicles of the synaptic bouton on the distal first order axon (b) react, whereas those of the first to second order (c) and of the second to third order (d) giant synapses do not. Such large differences in size and ZIO reactivity in populations of agranular vesicles have not to our knowledge been reported in other neuronal systems. In centres of invertebrate and vertebrate brains two size classes have been distinguishedl,6,sAL In the octopus brain both small and large agranular vesicles react with ZIO 1. The ZIO reactivity is used here as a histological criterion for distinguishing between synapses. Since synaptic vesicles of very different neuronal systems react positively with ZIO, this method cannot be associated with a specific transmitter substance 7. It appears, however, that agranular vesicles that do not react with Z I O are very rare. Two examples are the vesicles of contact (c) and (d) in the s q u i d 1 0 ; another example has been described by Dennison 3 in the rat olfactory bulb. Agranular synaptic vesicles are regularly found in synapses that contain Brain Research, 43 ( 1 9 7 2 ) 5 7 3 - 5 7 9
578
D. FROESCtt AND R. MARIIN
acetylcholine or amino acid as transmitter substance. But in cholinergic junctions, vesicles of very differing sizes have been described (frog neuromuscular junctione, 50 nm; rat diaphragm 14, 30-40 nm; Torpedo electric organ~, 80 nm). However, any comparison of vesicle size must be based on material treated in the same way and measured with a calibrated electron microscope. The chemical nature of the transmitter substance in the second to third order giant synapse of tile squid stellate ganglion (large agranular vesicles, 54 nm) is as yet unidentified 13, but most probably acetylcholine can be excluded (Miledi, personal communication). The other 4 synapses examined in the present work have not been studied physiologically. We cannot, therefore, associate the structural differences of the 5 synapses with different mechanisms of' transmission. On purely descriptive grounds we can state that in two subsequent contacts of this neuronal chain there are always two synapses with different synaptic vesicles. SUMMARY Four synaptic contacts in the squid giant fibre system (from the afferent boutons on the first order giant cell in the brain through the giant synapses in the paliiovisceral lobe and the stellate ganglion), as well as neuromuscular junctions in the fin, contain populations of agranular (electron transparent) synaptic vesicles. A classification of these vesicle populations is possible on the basis of the average vesicle diameter and the reactivity with zinc iodide-osmium (ZIO). We distinguish 4 synapses with significantly different average vesicle sizes, 3 synapses with ZIO-positive vesicles and two synapses with ZIO-negative vesicles. In two consecutive links of the giant fibre system there are always structurally different synaptic contacts. ACKNOWLEDGEMENT The support of D.F. by the Janggen-Poehn Foundation is gratefully acknowledged.
REFERENCES 1 BARLOW,J., AND MARTIN, R., Structural identification and distribution of synaptic profiles in the octopus brain using the zinc iodide-osmium method, Brain Research, 25 (1971) 241-253. 2 BIRKS,R., HUXLEY,H. E., AND KATZ, B., The fine structure of the neuromuscular junction of the frog, J. Physiol. (Lond.), 150 (1960) 134-144. 3 DENNISON,M. E., Electron stereoscopy as a means of classifying synaptic vesicles, J. Cell Sci., 8 (1971) 525-539. 4 GERVASIO,A., MARTIN,R., AND MIRALTO,A., Fine structure of synaptic contacts in the first order giant fibre system of the squid, Z. Zellforsch., 112 (1971) 85-96. 5 ISRAEL,M., GAUTRON,J., ET LESBETS,B., Fractionnement de l'organe 61ectrique de la Torpille: Localisation subcellulaire de l'ac6tylcholine, J. Neurochem., 17 (1970) 1441-1450. 6 JONES,D. G., The isolation of synaptic vesicles from octopus brain, Brain Research, 17 (1970) 181-193. 7 KAWAMA,E., AKERT,K., AND SANDRI,C., Zinc iodide-osmium tetroxide impregnation of nerve terminals in the spinal cord, Brain Research, 16 (1969) 325-331. Brain Research, 43 (1972) 573-579
SYNAPT1C VESICLES IN SQUID GIANT AXONS
579
8 LENN, N. J., AND REESE, T. S., The fine structure of nerve endings in the trapezoid body and the ventral cochlear nucleus, Amer. J. Anat., 118 (1966) 375-390. 9 MARTIN, R., AND BARLOW, J., Localisation of monoamines in nerves of the posterior salivary gland and salivary centre in the brain of octopus, Z. Zellforsch., 125 (1972) 16-30. 10 MARTIN, R., BARLOW,J., AND MIRALTO,A., Application of the zinc iodide-osmium tetroxide impregnation of synaptic vesicles in cephalopod nerves, Brain Research, 15 (1969) 1-16. II MARTIN, R., AND MILEDI, R., Effect of lanthanum ions on function and structure of the giant synapse of the squid, In preparation. 12 MCDONALD, n . M., AND RASMUSSEN,G. L., Association of acetylcholinesterase with one type of synaptic ending in the cochlear nucleus: an electron microscopy study, Anat. Rec., 163 (1969) 228. 13 MILEDI, R., Transmitter action in the giant synapse of the squid, Nature (Lond.), 223 (1969) 1284-1286. 14 PORTER, K. R., AND BONNEVILLE, M. A., Fine Structure of Cells and Tissues, Lea and Febiger, Philadelphia, 1968, pp. 165-166. 15 YOUNG, J. Z., Fused neurons and synaptic contacts in the giant nerve fibres of cephalopods, Phil. Trans. B, 229 (1939) 465-504.
Brain Research, 43 (1972) 573-579
573
HETEROGENEITY OF SYNAPTIC VESICLES IN THE SQUID GIANT FIBRE SYSTEM
D I E T E R FROESCH AND R A I N E R M A R T I N
Neurobiology Section, Stazione Zoologica di Napoli, Naples (Italy) (Accepted February 25th, 1972)
INTRODUCTION
The squid giant fibre system with its 3 sets of identified neurones4,15 offers the opportunity to compare synaptic fine structure in 4 successive contacts from the afferent input in the brain through two giant synapses in the palliovisceral lobe and the stellate ganglion. The most peripheral synapse in this chain, the neuromuscular junction between the third order giant axon and the mantle muscles, has not yet been identified. In the present study we examine the average vesicle size in populations of agranular vesicles in 5 synaptic contacts of the giant fibre system and in the fin musculature. The reactivity with zinc iodide-osmium (ZIO) is used as a further criterion to distinguish the synaptic vesicles of the 5 contacts. The results show that there are 4 significantly different sized classes of synaptic vesicles, that 3 of the vesicle populations react with ZIO, and that two do not. If these variations in vesicle size and reactivity reflect chemical differences between the synapses, one may postulate that in the giant fibre system there are 4 different mechanisms of synaptic transmission. At present the chemical nature of the transmitter substance has not been established in any of the contacts. MATERIAL AND METHODS
Specimens ofLoligo vulgaris were fixed by perfusion with 1% OsO4 in sea-water with 0.1 M cacodylate buffer (pH 7.4) or in a cephalopod Ringer solution9. Synapses (d) and (e) (see Results) were fixed by immersion of the stellate ganglion and fin muscles into 1.5~o OsOa dissolved in sea-water with 0.1 M cacodylate buffer. The vesicles of synapse (d) were measured from 3 animals of different mantle lengths (5, 12, 20 cm) that were fixed by perfusion or immersion. The vesicle diameters of this material are regularly distributed, as plotted in Fig. 2D. ZIO was applied as described in ref. 10~The tissues were dehydrated in ethanol and propylene oxide, and embedded in Durcupan. The sections were stained with lead citrate. The magnification of the electron microscope (Philips EM 200) was calibrated with a carbon replica of a diffrac-
Brain Research, 43 (1972) 573-579
574
D. F R O E S C t l A N D R. M A R T I N
tion grating (Ladd Research Inc.). The diameter o f the vesicles including the membrane was measured on electron micrographs × 88,000. In oval vesicles the smallest and the largest diameter were measured and the mean o f the two diameters was used for the statistics. The significance o f the size differences in the 5 vesicle populations was demonstrated by a t-test. RESULTS Fig. 1 represents schematically the connexions o f the giant fibre system o f the squid. The 5 synapses studied were: (a) afferent synaptic b o u t o n s on the cell b o d y o f the first order giant cell in the magnocellular lobe o f the brain4; (b) synaptic b o u t o n s on the distal first order giant axon in the palliovisceral lobe next to its contact with the second order giant axon4; (c) the axo-axonal contact o f the first order with the second order giant axon in the palliovisceral lobe4; (d) the axo-axonal contact o f the second order with the third order giant axon in the stellate ganglion11; and (e) neuromuscular junctions on muscle fibres o f the fin.
.U
a ,L
ax
%o
~x
a × II
~x
II
oOc~7~D
,~x III
e
Fig. 1. Left side, a schematic representation of the synaptic contacts in the giant fibre system; a, b, c, d and e are the synapses examined in this study (see text). I, II, III, first, second and third order giant neurones. Right side, a schematic drawing of the structural polarity of the 5 contacts (see text). I, first order giant cell; ax I, ax II, ax III, first, second and third order giant axons. Brain Research, 43 (1972) 573-579
575
SYNAPTIC VESICLES IN SQUID GIANT AXONS
o
lOO-i
m i
o 100
~0
0-
nn
-
in--m--
~
-I
d
27
J4
41
4~
,~S
62
~9
16
B~$
90
97
102 qr~ diameter
Fig. 2. Size distribution (nm) of vesicle diameter in the 5 synapses of Fig. 1. In b the vesicles inside the afferent bouton are represented. Contacts (a), (b), (d) and (e) have the structural characteristics of chemical synapses, i.e. a large synaptic cleft and masses of synaptic vesicles near the presynaptic membrane. Contact (c) shows the structural characteristics of chemical as well as of electrical synapses, the latter characterized by lack of synaptic vesicles and a gap junction of the membranes with a cleft smaller than 5 nm 4. While contacts (a), (c), (d) and (e) show accumulations of vesicles on one side only, contact (b) is characterized by masses of synaptic vesicles of different size on both sides of the synaptic membranes4. We do not have evidence that the neuromuscular contacts (e) are the terminals of giant axons. The structural polarity of the 5 contacts and the width of the synaptic clefts are schematically drawn in Fig. 1. The size distributions of the vesicles are shown in Fig. 2. The variation of vesicle size is highest in contact (c), but similar in contacts (a), (b), (d) and (e). The measured diameters of a larger number of synaptic vesicles in the 5 contacts are reported in Table I. The values are not significantly different between synapses (a) and (e), but between all the other synapses there are significant differences. Fig. 3 shows electron micrographs of vesicle populations of the 5 contacts not treated (left column) or treated (right column) with ZIO. The large majority of vesicles of contacts (c) and (d) do not react with ZIO, although a few vesicular profiles with reaction product are present. A control for the successful reaction consists in contact Brain Research, 43 (1972) 573-579
576
D. FROESCH ANI) R. MARTIN
Fig. 3. Electron m i c r o g r a p h s ( 7, 100,000) o f 5 vesicle populations in the synapses of Fig. 1. C o n t a c t b also c o n t a i n s vesicles on the side o f the first order axon, as s h o w n in c.
Brain Research, 43 (1972) 573-579
577
S Y N A P T I C VESICLES I N S Q U I D G I A N T A X O N S
TABLE
I
STATISTICS OF THE VESICLE DIAMETER OF THE 5 SYNAPSES
Synaptic contact
Number of vesicles
vesicle
Standard deviation:
Standard error:
measured: n
diameter:
s (nm)
s~ (nm)
Mean o f
(rim) a
369
42.5
5.1
0.014
b
427
51.2
6.6
0.015
c
610
69.1
12.8
0.021
d
601
54.4
5.2
0.008
e
499
43.5
6.9
0.014
Significance of the differences in means between two populations: t-test
a:b t a:c t a:d t a:e. t b:c t b:d t b:e t
= = = = = = = c:d t = C:C t = d :e, t =
21.5 P < 0.005 58.3 P < 0 . 0 0 5 73.4 P < 0.005 2.33 P = 0.01 39.7 P < 0.005 8.6 P < 0.005 17.6P < 0.005 38.5 P < 0.005 57.7 P < 0.005 30.3 P < 0 . 0 0 5
(b) in which a reactive and an unreactive population lie on either side of the same synaptic membrane pair 10. The population of large vesicles in contact (c) is identical with the population of vesicles on the side of the first order giant axon in contact (b). DISCUSSION
By comparison of the average size of 5 populations of agranular electrontransparent synaptic vesicles in 5 different synaptic contacts, 4 of which belong to identified neurones of the giant fibre system, we can distinguish 4 different size classes. In two populations the difference in means is probably not significant: the vesicles of afferent synaptic boutons on the first order giant neurone in the brain (a) and the vesicles of neuromuscular junctions in the fin (e). Both these populations react with ZIO. The 3 others, all with larger vesicles, belong to significantly different size classes. They are further distinguished by their reactivity with ZIO. The vesicles of the synaptic bouton on the distal first order axon (b) react, whereas those of the first to second order (c) and of the second to third order (d) giant synapses do not. Such large differences in size and ZIO reactivity in populations of agranular vesicles have not to our knowledge been reported in other neuronal systems. In centres of invertebrate and vertebrate brains two size classes have been distinguishedl,6,sAL In the octopus brain both small and large agranular vesicles react with ZIO 1. The ZIO reactivity is used here as a histological criterion for distinguishing between synapses. Since synaptic vesicles of very different neuronal systems react positively with ZIO, this method cannot be associated with a specific transmitter substance 7. It appears, however, that agranular vesicles that do not react with Z I O are very rare. Two examples are the vesicles of contact (c) and (d) in the s q u i d 1 0 ; another example has been described by Dennison 3 in the rat olfactory bulb. Agranular synaptic vesicles are regularly found in synapses that contain Brain Research, 43 ( 1 9 7 2 ) 5 7 3 - 5 7 9
578
D. FROESCtt AND R. MARIIN
acetylcholine or amino acid as transmitter substance. But in cholinergic junctions, vesicles of very differing sizes have been described (frog neuromuscular junctione, 50 nm; rat diaphragm 14, 30-40 nm; Torpedo electric organ~, 80 nm). However, any comparison of vesicle size must be based on material treated in the same way and measured with a calibrated electron microscope. The chemical nature of the transmitter substance in the second to third order giant synapse of tile squid stellate ganglion (large agranular vesicles, 54 nm) is as yet unidentified 13, but most probably acetylcholine can be excluded (Miledi, personal communication). The other 4 synapses examined in the present work have not been studied physiologically. We cannot, therefore, associate the structural differences of the 5 synapses with different mechanisms of' transmission. On purely descriptive grounds we can state that in two subsequent contacts of this neuronal chain there are always two synapses with different synaptic vesicles. SUMMARY Four synaptic contacts in the squid giant fibre system (from the afferent boutons on the first order giant cell in the brain through the giant synapses in the paliiovisceral lobe and the stellate ganglion), as well as neuromuscular junctions in the fin, contain populations of agranular (electron transparent) synaptic vesicles. A classification of these vesicle populations is possible on the basis of the average vesicle diameter and the reactivity with zinc iodide-osmium (ZIO). We distinguish 4 synapses with significantly different average vesicle sizes, 3 synapses with ZIO-positive vesicles and two synapses with ZIO-negative vesicles. In two consecutive links of the giant fibre system there are always structurally different synaptic contacts. ACKNOWLEDGEMENT The support of D.F. by the Janggen-Poehn Foundation is gratefully acknowledged.
REFERENCES 1 BARLOW,J., AND MARTIN, R., Structural identification and distribution of synaptic profiles in the octopus brain using the zinc iodide-osmium method, Brain Research, 25 (1971) 241-253. 2 BIRKS,R., HUXLEY,H. E., AND KATZ, B., The fine structure of the neuromuscular junction of the frog, J. Physiol. (Lond.), 150 (1960) 134-144. 3 DENNISON,M. E., Electron stereoscopy as a means of classifying synaptic vesicles, J. Cell Sci., 8 (1971) 525-539. 4 GERVASIO,A., MARTIN,R., AND MIRALTO,A., Fine structure of synaptic contacts in the first order giant fibre system of the squid, Z. Zellforsch., 112 (1971) 85-96. 5 ISRAEL,M., GAUTRON,J., ET LESBETS,B., Fractionnement de l'organe 61ectrique de la Torpille: Localisation subcellulaire de l'ac6tylcholine, J. Neurochem., 17 (1970) 1441-1450. 6 JONES,D. G., The isolation of synaptic vesicles from octopus brain, Brain Research, 17 (1970) 181-193. 7 KAWAMA,E., AKERT,K., AND SANDRI,C., Zinc iodide-osmium tetroxide impregnation of nerve terminals in the spinal cord, Brain Research, 16 (1969) 325-331. Brain Research, 43 (1972) 573-579
SYNAPT1C VESICLES IN SQUID GIANT AXONS
579
8 LENN, N. J., AND REESE, T. S., The fine structure of nerve endings in the trapezoid body and the ventral cochlear nucleus, Amer. J. Anat., 118 (1966) 375-390. 9 MARTIN, R., AND BARLOW, J., Localisation of monoamines in nerves of the posterior salivary gland and salivary centre in the brain of octopus, Z. Zellforsch., 125 (1972) 16-30. 10 MARTIN, R., BARLOW,J., AND MIRALTO,A., Application of the zinc iodide-osmium tetroxide impregnation of synaptic vesicles in cephalopod nerves, Brain Research, 15 (1969) 1-16. II MARTIN, R., AND MILEDI, R., Effect of lanthanum ions on function and structure of the giant synapse of the squid, In preparation. 12 MCDONALD, n . M., AND RASMUSSEN,G. L., Association of acetylcholinesterase with one type of synaptic ending in the cochlear nucleus: an electron microscopy study, Anat. Rec., 163 (1969) 228. 13 MILEDI, R., Transmitter action in the giant synapse of the squid, Nature (Lond.), 223 (1969) 1284-1286. 14 PORTER, K. R., AND BONNEVILLE, M. A., Fine Structure of Cells and Tissues, Lea and Febiger, Philadelphia, 1968, pp. 165-166. 15 YOUNG, J. Z., Fused neurons and synaptic contacts in the giant nerve fibres of cephalopods, Phil. Trans. B, 229 (1939) 465-504.
Brain Research, 43 (1972) 573-579