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Electron microscopic identification of cholinesterase-containing structures in the corpus striatum by diisopropyl fluorophosphate uptake
ESrERI>IEXTAI.
45, j41-j48
NEUROLOGY
(19i4)
Electron Microscopic Identification of CholinesteraseContaining Structures in the Corpus Striatum by Diisopropyl Fluorophosphate Uptake T. HATTORI Kirrsmot
Laboratory Vnivcrsity
oj
AND P. L. MCGEER
1
Nrwological Research, Department of of British Columbia, Vartcouvcv, Calzada Rcccivcd
July
Psychiatry,
27, 194
The binding of ‘H-diisopropyl fluorophosphate (DFP) to cholinesterase sites in the rat corpus striatum was studied by means of electron microscopic autoradiography. Small blocks of fixed tissue were incubated in the sequence nonradioactive DFP : Pyridine-Zaldoxime methiodide (2-PAM) : ‘H-DFP. The 2-PAM was used as a selective regenerating agent for cholinesterase sites prior to incubation with ‘I-I-DFP to label the sites. Terminal boutons had 227% of the grains and the highest relative grain density (RGD = % grains/o/o area) of 2.20. Dendrites and dendritic spines had 38% of the grains and a relative grain density of 1.41. Other structures, such as cell somata, axons, glial processes and blood vessels, had the remainder of the grains and a relative grain density between 0.58 and 0.66. Striatal boutons were divided into four types: asymmetrical groups 1 and 2, and symmetrical groups I and 2. One type-asymmetrical group l-was much more heavily labelled than the others, and was therefore presumed to be cholinergic. This type is densely packed with common round vesicles about 400-450 A in diameter and makes axospinous contacts.
INTRODUCTION Several studies (2, 6, 11-14) have indicated that the corpus striatum contains a high population of cholinergic interneurons. A developmental study of the rat neostriatum (5) showed a close correlation between the development of choline acetylase in the striatum and the appearance of common round vesicles asymmetrical axodendritic synapses containing which was taken as suggestive evidence of the morphological nature of cholinergic synapses. However the corpus striatum of the rat contains about 1 The excellent technical assistance of Mr. edged. This research was supported by a grant 541
Copyright 0 1974 by AcademicPress,Inc. AlI rights of reproduction in any form recerved
Nem Cvorkov is gratefully from the Province of British
acknowlColumbia.
542
HATTORI
AND
MCGEER
lo* dopaminergic nerve endings (l), and axoplasmic flow studies clearly show that these dopaminergic nerve endings also make asymmetrical synaptic contacts although they contain mildly pleomorphic vesicles (4). This paper reports a different approach to defining the nature of the cholinergic synapse through diisopropyl fluorophosphate (DFP) binding. DFP is a potent inhibitor of cholinesterase and carboxylic acid esterases (3). The acetylcholinesterase sites can be distinguished from other DFP reactive sites through the use of pyridine-Zaldoxime methiodide (Z-PAM). This latter agent selectively releases DFP from acetylcholinesterase binding sites (20, 21) but does not reverse the phosphate bond formed with other esterases. Thus, it is possible to pick out acetylcholinesterase sites by incubating brain tissue with nonradioactive DFP. reactivating the acetylcholinesterase sites with 2-PAM, labeling the vacated sites with radioactive DFP, and localizing the label by radioautography (15-18). Making lesions of known cholinergic axons causes a decrease in cholinesterase at the nerve endings indicating considerable presynaptic activity (9). At postsynaptic sites, such as the neuromuscular junction, acetylcholinesterase is also present in high concentrations (16, 17). Thus, DFP labelling should reveal something of the nature of both presynaptic and postsynaptic cholinergic elements. METHODS Three male Wistar rats were anesthetized with Nembutal (50 mg/kg) and then killed by aldehyde perfusion ( 1.5% glutaraldehyde in 0.06 M phosphate buffer, pH 7.4). The neostriatum was dissected out and cut into small cubes (1 mm3). Fourteen to 20 pieces were obtained from each neostriatum. Care was taken to collect samples from all parts of the nucleus. Small pieces were thoroughly rinsed in buffer for 2-3 hr and incubated in nonradioactive DFP ( 1O-3 M in 0.06 M phosphate buffer, pH 7.4) at room temperature for 20 min. This procedure was t’o phosphorylate all DFP-sensitive sites. The tissue blocks were then washed several times in buffer over a period of 20 min. They were then incubated in Z-PAM ( 10e3 M) at room temperature for 40 min to reactivate the acetylcholinesterase sites. After washing in buffer overnight, the blocks were incubated in 3HDFP (NEN 1O-4 M at 0.90 c/mmole) for 30 min at room temperature. Finally, they were rinsed in buffer, followed by nonradioactive DFP (10 min in 10e3 M, then 1 hr in 1e4 M) and buffer overnight. Tissue was postfixed in 1% buffered osmium tetroxide and embedded in epon-araldite. This sequence (nonradioactive DFP : 2-PAM : radioactive DFP) is known to introduce radioactivity only into DFP sensitive sites that have been reactivated by 2-PAM (15-18).
CHOLINESTERASE
IN
CORPUS
543
STRIATUM
Ilford L4 emulsion was coated by the loop method on gold sections from each block. After 4-6 weeks exposure, the sections were developed by Microdol X, fixed in Kodak Rapid Fix and stained by lead citrate. RESULTS Random pictures of the sections were taken on a Philips 201 electron microscope until a total of 248 silver grains had been obtained. A circle of 2,400 H, diameter was drawn around each grain on a photomicrograph. Assignment of the grain to a structure was in proportion to the area occupied by the structure within the circle. Small-magnification pictures were also randomly taken from the same grids to measure the relative over-all area which was occupied by each tissue structure (dendrite, axon, bouton, etc. ). The distribution of silver grains amongst the various structures of the neostriatum is summarized in Table 1. Terminal boutons had only 227% of the grains, but showed the highest relative grain density (i.e., the s grains/ s area) at 2.20. This was followed by the dendrites and/or spines which had 3S70 of the grains but a relative grain density of only 1.41. Other structures such as axons, cell somata, glia, and blood vessels all had a relative grain density of between 0.58 and 0.66 indicating nonpreferential labelling ( 19). Typical examples of the labelling pattern in terminal boutons and dendrites is shown in Fig 1A and B. The labelled boutons contain densely packed small round vesicles and make asymmertical synaptic contacts
TABLE OVER-ALL
DISTKIBUTION OF SILVER GKAINS AMONCC THE VAKIOUS STRUCTURES OF THE NEOSTKIATUM No.
Terminal bouton Dendrite & spine AXOlI Soma Blood vessel Glia Unidentified Total
1
(5’ /O
grain
Grain
54 95 50 27 7 11 4 248
22 38 20 11 3 4
Density (% grain/ 5%area) 492 1,244 1,452 1,007 250 349
10 27 30 21 5 7
2
100
4,794
100
2.20 1.41 0.66 0.53
0.50 0.57
544
HATTORI
AND
MCGEER
of terminal boutons and F ‘IG. 1. L ,abeling synapses containing labf :l in as ,ymmetrical x3 4.800.
dendrite common
by “ET-DFP. Note Iota .tiofl of round vesicles. A agni ficat ion
CIIOL1KESTER.ASE
1X
CORI’KS
STRIATUM
morpholagically different types of synapses FIG. 2. Four rat : A, asymmetrical group 1; B, asymmetrical group 2; D, symmetrical group 2. Magnification X 35,700.
545
in the neostriatum of the C, symmetrical group 1;
with small dendritic spines. The labeled dendrite shows the usual typical features. In the rat neostriatum sections, approximately SOY, of the total area occupied by boutons is occupied by asymmetrical synapses (Fig, 2). These asymmetrical synapses can be further subdivided into two groups. Group 1 contains common round vesicles of 400-450 A diameter, which are packed into the terminal zone. Most of these boutons make synaptic contacts with dendritic spines which usually contain a clear spinous apparatus. Group 2 contains slightly pleomorphic vesicles which are less densely packed than the first subgroup and which are distributed evenly over the terminal bouton. The size of vesicles is slightly larger than those of the first subgroup,
546
HATTORI
AND
MCGEER
ranging from 450-550 A in diameter. This type also typically makes axospinous synapses. A previous study (4) indicates group 2 represents dopaminergic terminals. This group is preferentially attacked by 6-hydroxydopamine, and is labeled by axoplasmic flow of radioactive protein from the substantia nigra. Symmetrical synapses constitute approximately 20% of the total bouton area (Fig. 2). Group 1 of the symmetrical synapses contains pleomorphic vesicles most of which are flattened. The vesicles are evenly distributed in the terminal bouton. This type usually synapses with major dendrites, although some axosomatic or axospinous synapsesare seen. Group 2 of the symmetrical type contains a few pleomorphic vesicles of different sizes in the region of the synaptic contact, but the rest of the terminal is empty. This classification and the relative percentages of the area in the rat neostriatum sections are quite similar to those provided by Kemp and Powell (7) for the cat caudate nucleus, although these workers did not recognize subgroups for asymmetrical synapses. When the distribution of 3H-DFP label was examined in these various types, it was found that 70% of the label in boutons was contained in asymmetrical group 1 terminals. This type had a relative grain density of 1.25 compared with 0.81 for asymmetrical group 2 and 0.50 for symmetrical synapses(Table 2). DISCUSSION The preferential labeling of presynaptic elements in the present study is in contrast to the experience with the neuromuscular junction. The primary site of the 3H-DFP binding at the endplate is postsynaptic and labelling of presynaptic elements has been attributed to radiation spread from the highly radioactive postsynaptic elements (18). In the corpus striatum, however, presynaptic elements are not invaginated in postsynaptic TABLE DISTRIBUTION
Asymmetrical Asymmetrical Symmetrical Symmetrical Total
group 1 group 2 group 1 group 2
2
OF SILVER GRAINS IN TERMINAL OF THE NEOSTRIATUM
BOUTONS
No.
%
grain
Grain
Area w
% Area
38 11
70 21
275 127
56 26
1.25 0.81
5
9
90
18
0.50
54
100
492
100
1.00
Density (% grain/ % ad
CIIOLINESTERASF.
IN
CORPUS
STRIATUM
547
receptors as they are at the neuromuscular junction. Therefore radiation spread from the postsynaptic sites would not be expected to distort the results, It is known that acetylcholinesterase is contained in ventral horn cells of the spinal cord (8) and is transported down peripheral nerves by axoplasmic flow ( 10). Therefore cholinesterase must exist presynaptically at the neuromuscular junction just as it does in the central nervous system. Data from the present study indicate it may be possible to identify presynaptic cholinesterase containing elements more readily in the central nervous system. It has been demonstrated that the postnatal development of choline acetylase shows a striking correlation with the development of asymmetrical round vesicle synapses in the neostriatum (5). In the present communication further evidence has been provided by a totally different procedure that this type of terminal bouton is cholinergic and most readily labeled by 3H-DFP. REFERENCES N. E., K. FUXE, B. HAMBERGER, and T. HBICFELT. 1966. A quantitative on the nigro-neostriatal dopamine neuron system in the rat. Actn Pltysiol.
1. AND~~N,
study
Stand. 67 : 306-312.
S. G., and L. L.
2. BUTCHER,
activity
in neostriatum.
BUTCHER.
BY&
1974. Origin
and modulation
of acetylcholine
Res. 71: 167-171.
3. DIXON,
M., and E. C. WEBB. 1964. “Enzymes.” Academic Press, New York. T., H. C. FIBIGER, P L. MCGEER, and L. MALEK. 1973. Analysis of the fine structure of the dopaminergic nigrostriatal projection by electron microscopic autoradiography. Exp. Nawol. 41 : 599-611.
4.
HATTORI,
5.
HATTORI,
T.,
E.rp. Newel. 6.
and
P. L.
MCGEER.
1973.
Synaptogenesis
in
the
corpus
striatum.
38 : 70-79.
T., P. L. MCGEER, H. C. FIBIGER, and E. G. MCGEER. 1973. On the of GABA-containing terminals in the substantia nigra. Electron microautoradiography and biochemical studies. Ijrair, Res. 54: 103-114. KEMP, J. M., and T. P. S. POWELL. 1971. The synaptic organization of the caudate nucleus. Phil. Trans. Roy. Sot. Lorld. Ser. B 262: 403-412. HATTORI,
source scopic
7.
8. KOELLE,
tral
G. B. 1954. The histochemical localization nervous system of the rat. J. Camp. Netlvol.
of cholinesterases 100: 211-235.
in the
cen-
9. LEWIS, P. R., and C. C. D. SHUTE. 1967. Confirmation from choline acetylase analysis of a massive cholinergic innervation to the rat hippocampus. J. Ph3uiol. (Lorldou) 191: 215-224. 10. LUBINSKA, L., and S. NIEMIERKO. 1971. Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves. Brain Res. 27 : 329-342. 11. LYNCH, G. S., P. A. LUCAS, and S. A. DEADWYLER. 1972. The demonstration of acetylcholinesterase containing neurons within the caudate nucleus of the rat. Brain Rex 45 : 617-627. 12. MCGEER, E. G., H. C. FIBIGER, P. L. MCGEER, and S. BROOKE. 1973. Temporal changes in amine synthesizing enzymes of rat extrapyramidal structures after hemitransections or 6-hydroxydopamine administration. Brain Rcs. 52 : 289-300.
548
HATTORI
AND
MCGEER
13. MCGEER, E. G., P. L. MCGEER, D. S. GREWAAL, and V. K. S~NGH. 1974. Striatal cholinergic interneurons and their relation to dopaminer,gic nerve endings. J. Pharwacologie (in the press). 14. MCGEER, P. L., E. G. MCGEER, H. C. FIBIGER. and V. WICKSON. 1971. Neostriatal choline acetylase and acetylcholinesterase following selective brain lesions. Brain Res. 35 : 308-314. 1.5. RODGERS, A. W., Z. DARZYNKIEWICZ, E. A. BARNARD, and M. M. SALPETER. 1966. Number and location of acetylcholinesterase molecules at motor endplates of the mouse. Natzrre London 210 : 1003-1006. 16. ROGERS, A. W., Z. DARZYNKIEWICZ, K. OSTROSKI., M. M. SALPETER, and E. A. BARNARD. 1969. Quantitative studies on enzymes in structures in striatal muscle by labelled inhibitor methods I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor endplates measured by radioautography. J. Cell Viol. 41: 665685. 17. SALPETER, M. M. 1967. Electron microscopic radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor endplates of a vertebrate twitch muscle. J. Cell Biol. 312: 379-389. 18. SALPETER, M. M. 1969. Electron microscopic radioautography as a quantitative tool in enzyme cytochemistry. II. The distribution of DFP-reactive sites at motor endplates of a vertebrate twitch muscle. J. Cell Biol. 42: 122-134. 19. SALPETER, M. M., and F. A. MCHENRY. 1973. Electron microscope autoradiography, pp. 113-152. 11%“Advanced Techniques in Biological Electron Microscopy.” J. K. Koehler [Ed.]. Springer-Verlag, New York. 20. WILSON, I. B. 1966. The inhibition and reactivation of acetylcholinesterase. Ants. N. Y. Acad. Sci. 135 : 177-183. 21. WILSON, I. B., S. GINSBURG, and C. QUAN. 1958. Molecular complementariness as basis for reactivation of alkyl phosphate-inhibited enzyme. Arch. Biochem. Biophys.
77 : 286-296.
45, j41-j48
NEUROLOGY
(19i4)
Electron Microscopic Identification of CholinesteraseContaining Structures in the Corpus Striatum by Diisopropyl Fluorophosphate Uptake T. HATTORI Kirrsmot
Laboratory Vnivcrsity
oj
AND P. L. MCGEER
1
Nrwological Research, Department of of British Columbia, Vartcouvcv, Calzada Rcccivcd
July
Psychiatry,
27, 194
The binding of ‘H-diisopropyl fluorophosphate (DFP) to cholinesterase sites in the rat corpus striatum was studied by means of electron microscopic autoradiography. Small blocks of fixed tissue were incubated in the sequence nonradioactive DFP : Pyridine-Zaldoxime methiodide (2-PAM) : ‘H-DFP. The 2-PAM was used as a selective regenerating agent for cholinesterase sites prior to incubation with ‘I-I-DFP to label the sites. Terminal boutons had 227% of the grains and the highest relative grain density (RGD = % grains/o/o area) of 2.20. Dendrites and dendritic spines had 38% of the grains and a relative grain density of 1.41. Other structures, such as cell somata, axons, glial processes and blood vessels, had the remainder of the grains and a relative grain density between 0.58 and 0.66. Striatal boutons were divided into four types: asymmetrical groups 1 and 2, and symmetrical groups I and 2. One type-asymmetrical group l-was much more heavily labelled than the others, and was therefore presumed to be cholinergic. This type is densely packed with common round vesicles about 400-450 A in diameter and makes axospinous contacts.
INTRODUCTION Several studies (2, 6, 11-14) have indicated that the corpus striatum contains a high population of cholinergic interneurons. A developmental study of the rat neostriatum (5) showed a close correlation between the development of choline acetylase in the striatum and the appearance of common round vesicles asymmetrical axodendritic synapses containing which was taken as suggestive evidence of the morphological nature of cholinergic synapses. However the corpus striatum of the rat contains about 1 The excellent technical assistance of Mr. edged. This research was supported by a grant 541
Copyright 0 1974 by AcademicPress,Inc. AlI rights of reproduction in any form recerved
Nem Cvorkov is gratefully from the Province of British
acknowlColumbia.
542
HATTORI
AND
MCGEER
lo* dopaminergic nerve endings (l), and axoplasmic flow studies clearly show that these dopaminergic nerve endings also make asymmetrical synaptic contacts although they contain mildly pleomorphic vesicles (4). This paper reports a different approach to defining the nature of the cholinergic synapse through diisopropyl fluorophosphate (DFP) binding. DFP is a potent inhibitor of cholinesterase and carboxylic acid esterases (3). The acetylcholinesterase sites can be distinguished from other DFP reactive sites through the use of pyridine-Zaldoxime methiodide (Z-PAM). This latter agent selectively releases DFP from acetylcholinesterase binding sites (20, 21) but does not reverse the phosphate bond formed with other esterases. Thus, it is possible to pick out acetylcholinesterase sites by incubating brain tissue with nonradioactive DFP. reactivating the acetylcholinesterase sites with 2-PAM, labeling the vacated sites with radioactive DFP, and localizing the label by radioautography (15-18). Making lesions of known cholinergic axons causes a decrease in cholinesterase at the nerve endings indicating considerable presynaptic activity (9). At postsynaptic sites, such as the neuromuscular junction, acetylcholinesterase is also present in high concentrations (16, 17). Thus, DFP labelling should reveal something of the nature of both presynaptic and postsynaptic cholinergic elements. METHODS Three male Wistar rats were anesthetized with Nembutal (50 mg/kg) and then killed by aldehyde perfusion ( 1.5% glutaraldehyde in 0.06 M phosphate buffer, pH 7.4). The neostriatum was dissected out and cut into small cubes (1 mm3). Fourteen to 20 pieces were obtained from each neostriatum. Care was taken to collect samples from all parts of the nucleus. Small pieces were thoroughly rinsed in buffer for 2-3 hr and incubated in nonradioactive DFP ( 1O-3 M in 0.06 M phosphate buffer, pH 7.4) at room temperature for 20 min. This procedure was t’o phosphorylate all DFP-sensitive sites. The tissue blocks were then washed several times in buffer over a period of 20 min. They were then incubated in Z-PAM ( 10e3 M) at room temperature for 40 min to reactivate the acetylcholinesterase sites. After washing in buffer overnight, the blocks were incubated in 3HDFP (NEN 1O-4 M at 0.90 c/mmole) for 30 min at room temperature. Finally, they were rinsed in buffer, followed by nonradioactive DFP (10 min in 10e3 M, then 1 hr in 1e4 M) and buffer overnight. Tissue was postfixed in 1% buffered osmium tetroxide and embedded in epon-araldite. This sequence (nonradioactive DFP : 2-PAM : radioactive DFP) is known to introduce radioactivity only into DFP sensitive sites that have been reactivated by 2-PAM (15-18).
CHOLINESTERASE
IN
CORPUS
543
STRIATUM
Ilford L4 emulsion was coated by the loop method on gold sections from each block. After 4-6 weeks exposure, the sections were developed by Microdol X, fixed in Kodak Rapid Fix and stained by lead citrate. RESULTS Random pictures of the sections were taken on a Philips 201 electron microscope until a total of 248 silver grains had been obtained. A circle of 2,400 H, diameter was drawn around each grain on a photomicrograph. Assignment of the grain to a structure was in proportion to the area occupied by the structure within the circle. Small-magnification pictures were also randomly taken from the same grids to measure the relative over-all area which was occupied by each tissue structure (dendrite, axon, bouton, etc. ). The distribution of silver grains amongst the various structures of the neostriatum is summarized in Table 1. Terminal boutons had only 227% of the grains, but showed the highest relative grain density (i.e., the s grains/ s area) at 2.20. This was followed by the dendrites and/or spines which had 3S70 of the grains but a relative grain density of only 1.41. Other structures such as axons, cell somata, glia, and blood vessels all had a relative grain density of between 0.58 and 0.66 indicating nonpreferential labelling ( 19). Typical examples of the labelling pattern in terminal boutons and dendrites is shown in Fig 1A and B. The labelled boutons contain densely packed small round vesicles and make asymmertical synaptic contacts
TABLE OVER-ALL
DISTKIBUTION OF SILVER GKAINS AMONCC THE VAKIOUS STRUCTURES OF THE NEOSTKIATUM No.
Terminal bouton Dendrite & spine AXOlI Soma Blood vessel Glia Unidentified Total
1
(5’ /O
grain
Grain
54 95 50 27 7 11 4 248
22 38 20 11 3 4
Density (% grain/ 5%area) 492 1,244 1,452 1,007 250 349
10 27 30 21 5 7
2
100
4,794
100
2.20 1.41 0.66 0.53
0.50 0.57
544
HATTORI
AND
MCGEER
of terminal boutons and F ‘IG. 1. L ,abeling synapses containing labf :l in as ,ymmetrical x3 4.800.
dendrite common
by “ET-DFP. Note Iota .tiofl of round vesicles. A agni ficat ion
CIIOL1KESTER.ASE
1X
CORI’KS
STRIATUM
morpholagically different types of synapses FIG. 2. Four rat : A, asymmetrical group 1; B, asymmetrical group 2; D, symmetrical group 2. Magnification X 35,700.
545
in the neostriatum of the C, symmetrical group 1;
with small dendritic spines. The labeled dendrite shows the usual typical features. In the rat neostriatum sections, approximately SOY, of the total area occupied by boutons is occupied by asymmetrical synapses (Fig, 2). These asymmetrical synapses can be further subdivided into two groups. Group 1 contains common round vesicles of 400-450 A diameter, which are packed into the terminal zone. Most of these boutons make synaptic contacts with dendritic spines which usually contain a clear spinous apparatus. Group 2 contains slightly pleomorphic vesicles which are less densely packed than the first subgroup and which are distributed evenly over the terminal bouton. The size of vesicles is slightly larger than those of the first subgroup,
546
HATTORI
AND
MCGEER
ranging from 450-550 A in diameter. This type also typically makes axospinous synapses. A previous study (4) indicates group 2 represents dopaminergic terminals. This group is preferentially attacked by 6-hydroxydopamine, and is labeled by axoplasmic flow of radioactive protein from the substantia nigra. Symmetrical synapses constitute approximately 20% of the total bouton area (Fig. 2). Group 1 of the symmetrical synapses contains pleomorphic vesicles most of which are flattened. The vesicles are evenly distributed in the terminal bouton. This type usually synapses with major dendrites, although some axosomatic or axospinous synapsesare seen. Group 2 of the symmetrical type contains a few pleomorphic vesicles of different sizes in the region of the synaptic contact, but the rest of the terminal is empty. This classification and the relative percentages of the area in the rat neostriatum sections are quite similar to those provided by Kemp and Powell (7) for the cat caudate nucleus, although these workers did not recognize subgroups for asymmetrical synapses. When the distribution of 3H-DFP label was examined in these various types, it was found that 70% of the label in boutons was contained in asymmetrical group 1 terminals. This type had a relative grain density of 1.25 compared with 0.81 for asymmetrical group 2 and 0.50 for symmetrical synapses(Table 2). DISCUSSION The preferential labeling of presynaptic elements in the present study is in contrast to the experience with the neuromuscular junction. The primary site of the 3H-DFP binding at the endplate is postsynaptic and labelling of presynaptic elements has been attributed to radiation spread from the highly radioactive postsynaptic elements (18). In the corpus striatum, however, presynaptic elements are not invaginated in postsynaptic TABLE DISTRIBUTION
Asymmetrical Asymmetrical Symmetrical Symmetrical Total
group 1 group 2 group 1 group 2
2
OF SILVER GRAINS IN TERMINAL OF THE NEOSTRIATUM
BOUTONS
No.
%
grain
Grain
Area w
% Area
38 11
70 21
275 127
56 26
1.25 0.81
5
9
90
18
0.50
54
100
492
100
1.00
Density (% grain/ % ad
CIIOLINESTERASF.
IN
CORPUS
STRIATUM
547
receptors as they are at the neuromuscular junction. Therefore radiation spread from the postsynaptic sites would not be expected to distort the results, It is known that acetylcholinesterase is contained in ventral horn cells of the spinal cord (8) and is transported down peripheral nerves by axoplasmic flow ( 10). Therefore cholinesterase must exist presynaptically at the neuromuscular junction just as it does in the central nervous system. Data from the present study indicate it may be possible to identify presynaptic cholinesterase containing elements more readily in the central nervous system. It has been demonstrated that the postnatal development of choline acetylase shows a striking correlation with the development of asymmetrical round vesicle synapses in the neostriatum (5). In the present communication further evidence has been provided by a totally different procedure that this type of terminal bouton is cholinergic and most readily labeled by 3H-DFP. REFERENCES N. E., K. FUXE, B. HAMBERGER, and T. HBICFELT. 1966. A quantitative on the nigro-neostriatal dopamine neuron system in the rat. Actn Pltysiol.
1. AND~~N,
study
Stand. 67 : 306-312.
S. G., and L. L.
2. BUTCHER,
activity
in neostriatum.
BUTCHER.
BY&
1974. Origin
and modulation
of acetylcholine
Res. 71: 167-171.
3. DIXON,
M., and E. C. WEBB. 1964. “Enzymes.” Academic Press, New York. T., H. C. FIBIGER, P L. MCGEER, and L. MALEK. 1973. Analysis of the fine structure of the dopaminergic nigrostriatal projection by electron microscopic autoradiography. Exp. Nawol. 41 : 599-611.
4.
HATTORI,
5.
HATTORI,
T.,
E.rp. Newel. 6.
and
P. L.
MCGEER.
1973.
Synaptogenesis
in
the
corpus
striatum.
38 : 70-79.
T., P. L. MCGEER, H. C. FIBIGER, and E. G. MCGEER. 1973. On the of GABA-containing terminals in the substantia nigra. Electron microautoradiography and biochemical studies. Ijrair, Res. 54: 103-114. KEMP, J. M., and T. P. S. POWELL. 1971. The synaptic organization of the caudate nucleus. Phil. Trans. Roy. Sot. Lorld. Ser. B 262: 403-412. HATTORI,
source scopic
7.
8. KOELLE,
tral
G. B. 1954. The histochemical localization nervous system of the rat. J. Camp. Netlvol.
of cholinesterases 100: 211-235.
in the
cen-
9. LEWIS, P. R., and C. C. D. SHUTE. 1967. Confirmation from choline acetylase analysis of a massive cholinergic innervation to the rat hippocampus. J. Ph3uiol. (Lorldou) 191: 215-224. 10. LUBINSKA, L., and S. NIEMIERKO. 1971. Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves. Brain Res. 27 : 329-342. 11. LYNCH, G. S., P. A. LUCAS, and S. A. DEADWYLER. 1972. The demonstration of acetylcholinesterase containing neurons within the caudate nucleus of the rat. Brain Rex 45 : 617-627. 12. MCGEER, E. G., H. C. FIBIGER, P. L. MCGEER, and S. BROOKE. 1973. Temporal changes in amine synthesizing enzymes of rat extrapyramidal structures after hemitransections or 6-hydroxydopamine administration. Brain Rcs. 52 : 289-300.
548
HATTORI
AND
MCGEER
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