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Cytochemical localization of acetylcholinesterase on isolated synaptosomes
SHORT COMMUNICATIONS
489
Cytochemical localization of acetylcholinesterase on isolated synaptosomes Acetylcholinesterase has been used in fractionation studies as a positive marker for cholinergic synaptosomes 1 and cholinergic synaptic plasma membranes 2. These investigations left open the question whether the enzyme was localized on the presynaptic, postsynaptic or on both membranes of the synaptic junctional complex. The work of Kasa and Csillik 4, using sections of cat cerebellar glomerulus, indicated that acetylcholinesterase was localized within the cleft of some but not all of the synapses in this region. However, even with these preparations, the precise localization of the enzyme on one or more membranes of the cleft remained uncertain. Since an unambiguous demonstration of either a pre- or postsynaptic origin of this marker enzyme would be most helpful in experiments designed to separate the two classes of membranes, we have undertaken studies with the aim of answering this question by cytochemical studies on isolated synaptosomes. Previous experiments 6 involving specific staining of such preparations indicated that this might be a fruitful approach. The cerebral cortex from ether anesthetized, female Sprague-Dawley rats (50-60 days old) was excised and immediately placed in ice-cold 0.32 M sucrose (pH 7.2-7.4). The homogenization and centrifugation conditions to obtain the crude mitochondrial fraction (P2) were based on the procedure of Gray and Whittaker 3 and has been previously described 6. The crude mitochondrial fraction was further resolved by isopycnic centrifugation in discontinuous sucrose gradients by the procedure of Gray and Whittaker 3 to yield a fraction rich in synaptosomes. This fraction was removed from the gradient with the aid of a syringe; it was diluted approximately two-fold with H20 and centrifuged at 74,000 × g to obtain a pellet which was resuspended in 2.0 ml of H20 (pH 7.2-7.4) and stored at --20 °C. The staining of the isolated synaptosomes for acetylcholinesterase was based on the procedure of Kasa and Csillik 4 (a complete description of the reagents used is given in their article). Aliquots were withdrawn from the suspension of synaptosomes and centrifuged at 76,000 × g for 40 min to obtain a small, tightly packed pellet. Test and control samples were withdrawn from the same stock suspension. The pellets were fixed in Ringer's medium containing 4 ~o formaldehyde for 1 h at 4-5 °C; they were then rinsed twice, first for 30 min and then for 5 min with 0.32 M sucrose at 4--5 °C. The pellets were reacted with the 'copper-lead-acetate' buffer containing acetylthiocholine for 1 h at 37 °C. The control pellet was pretreated for 10 min with the specific acetylcholinesterase inhibitor BW 284C514, 5 (provided by Burroughs, Wellcome and Co., Tuckahoe, N.Y.) prior to addition of acetylthiocholine. Next the pellets were rinsed twice, first for 30 min and then for 5 min with 0.32 M sucrose at 4-5 °C. Then 2.0 ml of Ringer's medium saturated with H2S were added; after 60 min at room temperature the pellet was rinsed twice with 0.32 M sucrose. The first rinse was left overnight at 4-5 °C and was followed by a second 5 min rinse. Samples were postfixed with 1 ~ OSO4, dehydrated and counterstained with uranyl acetate. Silver to gold sections were prepared and examined with a Hitachi Model HS7 electron microscope. Examinations of the control and test sections with the electron microscope revealed no apparent heavy staining in any particular structure of the control sections Brain Research, 41 (1972) 489-493
490
Brain Research, 41 (1972) 489-493
SHORT COMMUNICATIONS
SHORT COMMUNICATIONS
491
Fig. 1. Electron micrographs showing examples of localization of acetylcholinesterase (ACHE) activity on postsynaptic membrane. The nerve-ending particles (NEP) with post-synaptic membrane attached are indicated by ar, ows. An example of a postsynaptic membrane which did not stain for AChE is given in A. Examples of postsynaptic membranes which exhibited positive stains are given in B-D. The final magnifications are x 37,200, x 31,000, × 31,000, and × 34,000 for A - D , respectively.
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SHORT COMMUNICATIONS
(treated with BW 284C51). On the other hand, a very heavy staining of the postsynaptic membrane was readily apparent on the test sections (Fig. 1). In some cases a small amount of staining was observed on the presynaptic membrane but only in the region of the synaptic junction proper. Synaptosomes lacking an attached postsynaptic membrane did not show any apparent positive reaction on the presynaptic membrane. When seen, the intensity of staining observed in the presynaptic membrane was small compared to that observed on the attached postsynaptic membrane and could be a result of diffusion of a small amount of the thiocholine from its site of production on the postsynaptic side. Furthermore, not all attached postsynaptic membranes gave a positive reaction. It was not possible to obtain a precise estimate for the number of synapses which took up the stain because of the poor contrast of these sections (due to the fact that lead citrate staining was omitted). Only synapses which gave a positive acetylcholinesterase stain were easily observed; detection of other synapses was very difficult. No structure in these sections other than a synaptic complex was observed to give a positive reaction. These studies indicate that acetylcholinesterase is apparently concentrated on the postsynaptic membrane, a finding that is in agreement with observations reported for mouse muscle endplates 7. However, a strict interpretation of the results in terms of exclusive localization is not warranted since it is possible that the enzyme may be associated with other structures and may not have had access to the substrate, or, for whatever reason, may have remained undectected under the particular conditions used, for instance because of being present in insufficiently high concentrations. This investigation formed part of the Brain Research Program directed by Profs. H. R. Mahler and W. J. Moore, supported by research grants from the National Science Foundation (No. GB17157) and from the National Institute of Neurological Diseases and Stroke, N.I.H., U.S. Dept. of Health, Education and Welfare (NS 08309). Section of Neurobiology, The Institute of Psychiatric Research, and Department of Psychiatry, Indiana University Medical Center, Indianapolis, lnd. 46202 (U.S.A.) and Brain Research Group, Department of Chemistry, Indiana University, Bloomington, Ind. 47401 (U.S.A.)
W. J. McBRIDE
HAROLD COHEN
1 DEROBERTIS,E., PELLEGRINODEIRALDI,A., RODRIGUEZDE LORESARNAIZ,G., ANDSALGANICOFF,
L., Cholinergic and noncholinergic nerve endings in rat brain. I. Isolation and subceltutar distribution of acetylcholine and acetylcholinesterase, J. Neurochem., 9 (1962) 23-35. 2 DEROBERTIS,E., RODRIGUEZDE LORESARNAIZ,G., SALGANICOFF,L., PELLEGRINODE IRALDI,A., AND ZIEHER, L. M., Isolation of synaptic vesicles and structural organization of the acetylcholine system within brain nerve endings, J. Neurochem., 10 (1963) 225-235. 3 GRAY, E. G., AND WHITTAKER, V. P., The isolation of nerve endings from brain: An electronmicroscopic study ofcell fragments derived by homogenization and centrifugation, J. Anat. (Lond), 96 (1962) 79-88. Brain Research, 41 (1972) 489-493
SHORT COMMUNICATIONS
493
4 KASA, P., AND CSILLIK,B., Electron microscopic localization of cholinesterase by a copper-leadthiocholine technique, J. Neurochem., 13 (1966) 1345-1349. 5 KLINGMAN, G. I., KLINGMAN, J. D., AND POLISZCZUK, A., Acetyl- and pseudocholinesterase activities in sympathetic ganglia of rats, J. Neurochem., 15 (1968) 1121-1130. 6 McBRIDE, W. J., MAHLER,H. R., MOORE, W. J., AND WHITE, F. P., Isolation and characterization of membranes from rat cerebral cortex, J. Neurobiol., 2 (1970) 73-92. 7 SALPETER, M. M., Electron microscope 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 (1969) 122-134. (Accepted March 22nd, 1972)
Brain Research, 41 (1972) 489-493
489
Cytochemical localization of acetylcholinesterase on isolated synaptosomes Acetylcholinesterase has been used in fractionation studies as a positive marker for cholinergic synaptosomes 1 and cholinergic synaptic plasma membranes 2. These investigations left open the question whether the enzyme was localized on the presynaptic, postsynaptic or on both membranes of the synaptic junctional complex. The work of Kasa and Csillik 4, using sections of cat cerebellar glomerulus, indicated that acetylcholinesterase was localized within the cleft of some but not all of the synapses in this region. However, even with these preparations, the precise localization of the enzyme on one or more membranes of the cleft remained uncertain. Since an unambiguous demonstration of either a pre- or postsynaptic origin of this marker enzyme would be most helpful in experiments designed to separate the two classes of membranes, we have undertaken studies with the aim of answering this question by cytochemical studies on isolated synaptosomes. Previous experiments 6 involving specific staining of such preparations indicated that this might be a fruitful approach. The cerebral cortex from ether anesthetized, female Sprague-Dawley rats (50-60 days old) was excised and immediately placed in ice-cold 0.32 M sucrose (pH 7.2-7.4). The homogenization and centrifugation conditions to obtain the crude mitochondrial fraction (P2) were based on the procedure of Gray and Whittaker 3 and has been previously described 6. The crude mitochondrial fraction was further resolved by isopycnic centrifugation in discontinuous sucrose gradients by the procedure of Gray and Whittaker 3 to yield a fraction rich in synaptosomes. This fraction was removed from the gradient with the aid of a syringe; it was diluted approximately two-fold with H20 and centrifuged at 74,000 × g to obtain a pellet which was resuspended in 2.0 ml of H20 (pH 7.2-7.4) and stored at --20 °C. The staining of the isolated synaptosomes for acetylcholinesterase was based on the procedure of Kasa and Csillik 4 (a complete description of the reagents used is given in their article). Aliquots were withdrawn from the suspension of synaptosomes and centrifuged at 76,000 × g for 40 min to obtain a small, tightly packed pellet. Test and control samples were withdrawn from the same stock suspension. The pellets were fixed in Ringer's medium containing 4 ~o formaldehyde for 1 h at 4-5 °C; they were then rinsed twice, first for 30 min and then for 5 min with 0.32 M sucrose at 4--5 °C. The pellets were reacted with the 'copper-lead-acetate' buffer containing acetylthiocholine for 1 h at 37 °C. The control pellet was pretreated for 10 min with the specific acetylcholinesterase inhibitor BW 284C514, 5 (provided by Burroughs, Wellcome and Co., Tuckahoe, N.Y.) prior to addition of acetylthiocholine. Next the pellets were rinsed twice, first for 30 min and then for 5 min with 0.32 M sucrose at 4-5 °C. Then 2.0 ml of Ringer's medium saturated with H2S were added; after 60 min at room temperature the pellet was rinsed twice with 0.32 M sucrose. The first rinse was left overnight at 4-5 °C and was followed by a second 5 min rinse. Samples were postfixed with 1 ~ OSO4, dehydrated and counterstained with uranyl acetate. Silver to gold sections were prepared and examined with a Hitachi Model HS7 electron microscope. Examinations of the control and test sections with the electron microscope revealed no apparent heavy staining in any particular structure of the control sections Brain Research, 41 (1972) 489-493
490
Brain Research, 41 (1972) 489-493
SHORT COMMUNICATIONS
SHORT COMMUNICATIONS
491
Fig. 1. Electron micrographs showing examples of localization of acetylcholinesterase (ACHE) activity on postsynaptic membrane. The nerve-ending particles (NEP) with post-synaptic membrane attached are indicated by ar, ows. An example of a postsynaptic membrane which did not stain for AChE is given in A. Examples of postsynaptic membranes which exhibited positive stains are given in B-D. The final magnifications are x 37,200, x 31,000, × 31,000, and × 34,000 for A - D , respectively.
492
SHORT COMMUNICATIONS
(treated with BW 284C51). On the other hand, a very heavy staining of the postsynaptic membrane was readily apparent on the test sections (Fig. 1). In some cases a small amount of staining was observed on the presynaptic membrane but only in the region of the synaptic junction proper. Synaptosomes lacking an attached postsynaptic membrane did not show any apparent positive reaction on the presynaptic membrane. When seen, the intensity of staining observed in the presynaptic membrane was small compared to that observed on the attached postsynaptic membrane and could be a result of diffusion of a small amount of the thiocholine from its site of production on the postsynaptic side. Furthermore, not all attached postsynaptic membranes gave a positive reaction. It was not possible to obtain a precise estimate for the number of synapses which took up the stain because of the poor contrast of these sections (due to the fact that lead citrate staining was omitted). Only synapses which gave a positive acetylcholinesterase stain were easily observed; detection of other synapses was very difficult. No structure in these sections other than a synaptic complex was observed to give a positive reaction. These studies indicate that acetylcholinesterase is apparently concentrated on the postsynaptic membrane, a finding that is in agreement with observations reported for mouse muscle endplates 7. However, a strict interpretation of the results in terms of exclusive localization is not warranted since it is possible that the enzyme may be associated with other structures and may not have had access to the substrate, or, for whatever reason, may have remained undectected under the particular conditions used, for instance because of being present in insufficiently high concentrations. This investigation formed part of the Brain Research Program directed by Profs. H. R. Mahler and W. J. Moore, supported by research grants from the National Science Foundation (No. GB17157) and from the National Institute of Neurological Diseases and Stroke, N.I.H., U.S. Dept. of Health, Education and Welfare (NS 08309). Section of Neurobiology, The Institute of Psychiatric Research, and Department of Psychiatry, Indiana University Medical Center, Indianapolis, lnd. 46202 (U.S.A.) and Brain Research Group, Department of Chemistry, Indiana University, Bloomington, Ind. 47401 (U.S.A.)
W. J. McBRIDE
HAROLD COHEN
1 DEROBERTIS,E., PELLEGRINODEIRALDI,A., RODRIGUEZDE LORESARNAIZ,G., ANDSALGANICOFF,
L., Cholinergic and noncholinergic nerve endings in rat brain. I. Isolation and subceltutar distribution of acetylcholine and acetylcholinesterase, J. Neurochem., 9 (1962) 23-35. 2 DEROBERTIS,E., RODRIGUEZDE LORESARNAIZ,G., SALGANICOFF,L., PELLEGRINODE IRALDI,A., AND ZIEHER, L. M., Isolation of synaptic vesicles and structural organization of the acetylcholine system within brain nerve endings, J. Neurochem., 10 (1963) 225-235. 3 GRAY, E. G., AND WHITTAKER, V. P., The isolation of nerve endings from brain: An electronmicroscopic study ofcell fragments derived by homogenization and centrifugation, J. Anat. (Lond), 96 (1962) 79-88. Brain Research, 41 (1972) 489-493
SHORT COMMUNICATIONS
493
4 KASA, P., AND CSILLIK,B., Electron microscopic localization of cholinesterase by a copper-leadthiocholine technique, J. Neurochem., 13 (1966) 1345-1349. 5 KLINGMAN, G. I., KLINGMAN, J. D., AND POLISZCZUK, A., Acetyl- and pseudocholinesterase activities in sympathetic ganglia of rats, J. Neurochem., 15 (1968) 1121-1130. 6 McBRIDE, W. J., MAHLER,H. R., MOORE, W. J., AND WHITE, F. P., Isolation and characterization of membranes from rat cerebral cortex, J. Neurobiol., 2 (1970) 73-92. 7 SALPETER, M. M., Electron microscope 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 (1969) 122-134. (Accepted March 22nd, 1972)
Brain Research, 41 (1972) 489-493