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The negative staining ofOctopus synaptosomes
378
SHORT COMMUNICATIONS
The negative staining of Octopus synaptosomes Negative staining has proved a very useful ancillary technique in morphological investigations of subcellular fractions of nervous tissue2,4,10,11. Besides serving as a rapid means of demonstrating the structures present in a fraction, it has provided information concerning the morphology of synaptosomes and especially of isolated synaptic vesicles 11, additional to that obtained from positively stained, sectioned material. While this has been true of mammalian tissues, fractionation studies of Octopus brain have been circumscribed by the lack of a satisfactory negative staining technique in the presence of sucrose with a molarity as high as 1 M (ref. 5). Florey and Winesdorfer 3, in their study of Octopus brain fractions, used a 1 : 1 mixture of saline and 1 M sucrose as the homogenization medium and performed no negative staining. Sheridan et al. 9 were successful in negatively staining fractions of"the electric organ of Torpedo, but these had been homogenized in 0.5 M sucrose containing 0.33 M urea. Two experimental approaches aimed at overcoming the difficulty with negative staining in Octopus material are suggested in the present paper. In the first case homogenization was carried out in 0.8 M sucrose which, from a morphological standpoint, gives equally good results as a medium with a final molarity of 1 M (ref. 5). The corresponding negative staining was with ammonium molybdate. In the second case homogenization was in 1.1 M glucose 1 and the negative staining was with phosphotungstic acid (PTA). In the first part of the study synaptosomes were prepared from the supraoesophageal lobes of the brain of Octopus vulgaris by gentle manual homogenization in 0.8 M sucrose, followed by centrifugation at 5,000 × g for 15 min to remove large cellular debris. The resulting supernatant was used as the source of the synaptosomes examined in the study, and it was treated in one of two ways. It was either mixed with an equal volume of ammonium molybdate and a drop of the mixture was placed on a grid, or it was dropped on a grid alone, largely removed by filter paper and followed by a drop of ammonium molybdate. In either case, the solution on the grid was drained off after about 1 min with filter paper. A 6 ~ solution of ammonium molybdate was employed in order that the concentration of the stain would have an osmolality equivalent to that of the sucrose (based on the technique of Muscatello and HorneS). The pH of the ammonium molybdate was adjusted to 7.3, no fixative was used, and all procedures were carried out at 0-4°C. In the second part of the study synaptosomes were prepared from the optic lobes of the brain of Octopus vulgaris as described by Jones 5, except that the tissue was homogenized in 1.1 M glucose. The resulting 10~o (w/v) suspension was centrifuged for 11 rain at 1,000 × g to give the P1 pellet. The supernatant was centrifuged at 17,000 × g for 1 h, giving the P2 fraction. Both fractions contain synaptosomes 5 and both were used as sources of synaptosomes in the present experiment. For negative staining, each pellet was resuspended in about 2 ml of 1.1 M glucose and a drop of the suspension placed on a grid for 1 min. Most of this was carefully drained offwith filter paper and a drop of 1 ~ PTA in ethanol (pH 7.3) was added for 1 min.
Brain Research, 29 (1971) 378-382
Figs. 1 and 2. Synaptic vesicles (sv) of Varying shapes and characteristics, including elliptical vesicles (ev), are present in these synaptosomes. The limiting membrane of many vesicles (arrows) is clearly seen, and areas of intense contrast (ic) are found in some of them. The contact region between one of the postsynaptic processes (p) and the synaptosome in Fig. 1 has cleft material (cm) running parallel to the pre- and postsynaptic membranes. Negatively stained with ammonium molybdate. Fig. 1, x 70,000; Fig. 2, x 52,500.
380
SHORT COMMUNICATIONS
In both parts of the experiment the grids, once dry, were examined with a Siemens Elmiskop I electron microscope at primary magnifications of × 15,000 to x 20,000 in most instances. When ammonium molybdate is used for negative staining, the synaptic vesicles within synaptosomes are usually readily visualized (Figs. 1, 2). Their limiting membranes are clearly demarcated due to the penetration of the stain, and their appearance resembles that described by Whittaker and Sheridan 11 for vesicles resuspended in sodium phosphate buffer prior to negative staining with PTA. The thickness of the clearly defined bilayered limiting membranes is of the order of 8-11 nm, although considerable variations occur especially in those instances where the membranes appear to be pulled apart at one pole of the vesicle to form elliptical vesicles. Areas of intense contrast are present in association with the limiting membranes of a few vesicles (Fig. 2). Whittaker and Sheridan 11 considered that such areas represent 'lipped' vesicles or solid material deposited within the vesicle. The majority of synaptic vesicles have diameters between 60 and 95 nm. The mean value for negatively stained vesicles in the present study is 75-85 nm, and therefore higher than the corresponding figure for vesicles in embedded and sectioned tissue. A similar phenomenon was observed by De Robertis et al. ~ who noted that the negative staining of vesicles isolated from rat brain gives dimensions about 25 larger than those characteristic of embedded vesicles.
Fig. 3. Large granular vesicles (lgv) are seen within synaptosomes, the background of which consists of a cytoplasmic framework (cf). Negatively stained with PTA. x 43,000. Brain Research,
29 (1971) 378-382
SHORT COMMUNICATIONS
381
Postsynaptic processes are seen adhering to the presynaptic components o f synaptosomes in Fig. 1. Little definition of structures within the processes is possible, and little detail can be made out of material within the cleft. However, a band or plate orientated parallel to the pre- and postsynaptic membranes is present in the cleft. PTA, used in conjunction with glucose homogenization, presents a different picture of the internal constitution of synaptosomes (Fig. 3). Agranular synaptic vesicles are generally poorly defined, although large granular vesicles are prominent. The cytoplasm may be occupied by a framework in which there are 'spaces', some of which are regular and spherical. The possibility arises that some at least of these spaces may be occupied by synaptic vesicles, as many of them are approximately 80 nm in diameter. The present study demonstrates the possibility of obtaining satisfactory micrographs of negatively stained tissue derived from Octopus brain. Either sucrose or glucose can be used as homogenizing media, although sucrose tends to give inconsistent results at a molarity as high as 0.8 M. A number of experiments in the present series were unsuccessful due to excessive sucrose contamination, and further work is required to devise a consistently satisfactory method of negative staining for Octopus nervous tissue. As can be seen from Fig. 1, the intimate relationship between the pre- and postsynaptic components of synaptosomes may be maintained, and cleft material may be recognizable at the contact region. Unfortunately, only a minimal amount of detailed structure is visible, thereby rendering negative staining in its present form of limited value in investigations of synaptic ultrastructure. In spite of this, it is possible that the cleft material corresponds to one of the entities of the contact region described in octopus embedded material, namely the intermediate band s or synaptic plate 7. Under the conditions employed in the present investigation, ammonium molybdate has proved a satisfactory stain for the investigation of the ultrastructure of synaptic vesicles. This accords with the conclusions of Muscatello and Hornes who showed that the membrane organization of red blood cells, mitochondria and microsomes was well preserved in the presence of ammonium molybdate. PTA, in unfixed material, appears not to stain the agranular vesicles as such but the cytoplasmic framework in which they are embedded. It is possible that the vesicles are situated in the spaces of this framework, although great care must be taken in any interpretation of this nature. However, if this is so, we may have here a means of highlighting the cytoplasmic framework by apparently 'removing' the vesicles. I would like to thank Professor D. B. Allbrook for reading the manuscript, and Mr. D, Stewart for photographic assistance.
Department of Anatomy, University of Western Australia, Nedlands, W.A. 6009 (Australia)
D. G. JONES
Brain Research, 29 (1971) 378-382
382
SHORT COMMUNICATIONS
1 COTTRELL, G. A., Separation and properties of subcellular particles associated with 5-hydroxytryptamine, with acetylcholine and with an unidentified cardio-excitatory substance from Mercenaria nervous tissue, Comp. Biochem. Physiol., 17 (1966) 891-907. 2 DE ROBERTIS, E., RODRIGUEZ DE LORES ARNAIZ, G., SALGANICOFF,L., PEELEGRINO DE IRALDI, A., AND ZIEHER, L. M., Isolation of synaptic vesicles and structural organization of the acetylcholine system within brain nerve endings, J. Neurochem., l0 (1963) 225-235. 3 FLOREY, E., AND WINESDOREER, J., Cholinergic nerve endings in octopus brain, J. Neurochem., 15 (1968) 169-177. 4 HORNE, R. W., AND WHITTAKER,V. P., The use of the negative staining method for the electronmicroscopic study of subcellular particles from animal tissues, Z. Zellforsch., 58 (1962) 1-16. 5 JONES, D. G., An electron-microscope study of subcellular fractions of Octopus brain, J. Cell Sci., 2 (1967) 573-586. 6 JONES, O. G., The fine structure of the synaptic membrane adhesions on octopus synaptosomes, Z. Zellforsch., 88 (1968) 457469. 7 JONES, D. G., A further contribution to the study of the contact region of Octopus synaptosomes, Z. Zellforsch., 103 (1970) 48-60. 8 MUSCATELLO, U., AND HORNE, R. W., Effect of the tonicity of some negative-staining solutions on the elementary structure of membrane-bounded systems, J. Ultrastruct. Res., 25 (1968) 73-83. 9 SHERIDAN,M. N., WHITTAKER, V. P., AND ISRAEL,M., The subcellular fractionation of the electric organ of Torpedo, Z. Zellforsch., 74 (1966) 291-307. 10 WHITTAKER, V. P., MICHAELSON, I. A., AND KIRKLAND, R. J. A., The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'), Biochem. J., 90 (I 964) 293-303. 11 WHITTAKER, V. P., AND SHERIDAN, M. N., The morphology and acetylcholine content of isolated cerebral cortical synaptic vesicles, J. Neurochem., 12 (•965) 363-372.
(Accepted April 2nd, 1971)
Brain Research, 29 (1971) 378-382
SHORT COMMUNICATIONS
The negative staining of Octopus synaptosomes Negative staining has proved a very useful ancillary technique in morphological investigations of subcellular fractions of nervous tissue2,4,10,11. Besides serving as a rapid means of demonstrating the structures present in a fraction, it has provided information concerning the morphology of synaptosomes and especially of isolated synaptic vesicles 11, additional to that obtained from positively stained, sectioned material. While this has been true of mammalian tissues, fractionation studies of Octopus brain have been circumscribed by the lack of a satisfactory negative staining technique in the presence of sucrose with a molarity as high as 1 M (ref. 5). Florey and Winesdorfer 3, in their study of Octopus brain fractions, used a 1 : 1 mixture of saline and 1 M sucrose as the homogenization medium and performed no negative staining. Sheridan et al. 9 were successful in negatively staining fractions of"the electric organ of Torpedo, but these had been homogenized in 0.5 M sucrose containing 0.33 M urea. Two experimental approaches aimed at overcoming the difficulty with negative staining in Octopus material are suggested in the present paper. In the first case homogenization was carried out in 0.8 M sucrose which, from a morphological standpoint, gives equally good results as a medium with a final molarity of 1 M (ref. 5). The corresponding negative staining was with ammonium molybdate. In the second case homogenization was in 1.1 M glucose 1 and the negative staining was with phosphotungstic acid (PTA). In the first part of the study synaptosomes were prepared from the supraoesophageal lobes of the brain of Octopus vulgaris by gentle manual homogenization in 0.8 M sucrose, followed by centrifugation at 5,000 × g for 15 min to remove large cellular debris. The resulting supernatant was used as the source of the synaptosomes examined in the study, and it was treated in one of two ways. It was either mixed with an equal volume of ammonium molybdate and a drop of the mixture was placed on a grid, or it was dropped on a grid alone, largely removed by filter paper and followed by a drop of ammonium molybdate. In either case, the solution on the grid was drained off after about 1 min with filter paper. A 6 ~ solution of ammonium molybdate was employed in order that the concentration of the stain would have an osmolality equivalent to that of the sucrose (based on the technique of Muscatello and HorneS). The pH of the ammonium molybdate was adjusted to 7.3, no fixative was used, and all procedures were carried out at 0-4°C. In the second part of the study synaptosomes were prepared from the optic lobes of the brain of Octopus vulgaris as described by Jones 5, except that the tissue was homogenized in 1.1 M glucose. The resulting 10~o (w/v) suspension was centrifuged for 11 rain at 1,000 × g to give the P1 pellet. The supernatant was centrifuged at 17,000 × g for 1 h, giving the P2 fraction. Both fractions contain synaptosomes 5 and both were used as sources of synaptosomes in the present experiment. For negative staining, each pellet was resuspended in about 2 ml of 1.1 M glucose and a drop of the suspension placed on a grid for 1 min. Most of this was carefully drained offwith filter paper and a drop of 1 ~ PTA in ethanol (pH 7.3) was added for 1 min.
Brain Research, 29 (1971) 378-382
Figs. 1 and 2. Synaptic vesicles (sv) of Varying shapes and characteristics, including elliptical vesicles (ev), are present in these synaptosomes. The limiting membrane of many vesicles (arrows) is clearly seen, and areas of intense contrast (ic) are found in some of them. The contact region between one of the postsynaptic processes (p) and the synaptosome in Fig. 1 has cleft material (cm) running parallel to the pre- and postsynaptic membranes. Negatively stained with ammonium molybdate. Fig. 1, x 70,000; Fig. 2, x 52,500.
380
SHORT COMMUNICATIONS
In both parts of the experiment the grids, once dry, were examined with a Siemens Elmiskop I electron microscope at primary magnifications of × 15,000 to x 20,000 in most instances. When ammonium molybdate is used for negative staining, the synaptic vesicles within synaptosomes are usually readily visualized (Figs. 1, 2). Their limiting membranes are clearly demarcated due to the penetration of the stain, and their appearance resembles that described by Whittaker and Sheridan 11 for vesicles resuspended in sodium phosphate buffer prior to negative staining with PTA. The thickness of the clearly defined bilayered limiting membranes is of the order of 8-11 nm, although considerable variations occur especially in those instances where the membranes appear to be pulled apart at one pole of the vesicle to form elliptical vesicles. Areas of intense contrast are present in association with the limiting membranes of a few vesicles (Fig. 2). Whittaker and Sheridan 11 considered that such areas represent 'lipped' vesicles or solid material deposited within the vesicle. The majority of synaptic vesicles have diameters between 60 and 95 nm. The mean value for negatively stained vesicles in the present study is 75-85 nm, and therefore higher than the corresponding figure for vesicles in embedded and sectioned tissue. A similar phenomenon was observed by De Robertis et al. ~ who noted that the negative staining of vesicles isolated from rat brain gives dimensions about 25 larger than those characteristic of embedded vesicles.
Fig. 3. Large granular vesicles (lgv) are seen within synaptosomes, the background of which consists of a cytoplasmic framework (cf). Negatively stained with PTA. x 43,000. Brain Research,
29 (1971) 378-382
SHORT COMMUNICATIONS
381
Postsynaptic processes are seen adhering to the presynaptic components o f synaptosomes in Fig. 1. Little definition of structures within the processes is possible, and little detail can be made out of material within the cleft. However, a band or plate orientated parallel to the pre- and postsynaptic membranes is present in the cleft. PTA, used in conjunction with glucose homogenization, presents a different picture of the internal constitution of synaptosomes (Fig. 3). Agranular synaptic vesicles are generally poorly defined, although large granular vesicles are prominent. The cytoplasm may be occupied by a framework in which there are 'spaces', some of which are regular and spherical. The possibility arises that some at least of these spaces may be occupied by synaptic vesicles, as many of them are approximately 80 nm in diameter. The present study demonstrates the possibility of obtaining satisfactory micrographs of negatively stained tissue derived from Octopus brain. Either sucrose or glucose can be used as homogenizing media, although sucrose tends to give inconsistent results at a molarity as high as 0.8 M. A number of experiments in the present series were unsuccessful due to excessive sucrose contamination, and further work is required to devise a consistently satisfactory method of negative staining for Octopus nervous tissue. As can be seen from Fig. 1, the intimate relationship between the pre- and postsynaptic components of synaptosomes may be maintained, and cleft material may be recognizable at the contact region. Unfortunately, only a minimal amount of detailed structure is visible, thereby rendering negative staining in its present form of limited value in investigations of synaptic ultrastructure. In spite of this, it is possible that the cleft material corresponds to one of the entities of the contact region described in octopus embedded material, namely the intermediate band s or synaptic plate 7. Under the conditions employed in the present investigation, ammonium molybdate has proved a satisfactory stain for the investigation of the ultrastructure of synaptic vesicles. This accords with the conclusions of Muscatello and Hornes who showed that the membrane organization of red blood cells, mitochondria and microsomes was well preserved in the presence of ammonium molybdate. PTA, in unfixed material, appears not to stain the agranular vesicles as such but the cytoplasmic framework in which they are embedded. It is possible that the vesicles are situated in the spaces of this framework, although great care must be taken in any interpretation of this nature. However, if this is so, we may have here a means of highlighting the cytoplasmic framework by apparently 'removing' the vesicles. I would like to thank Professor D. B. Allbrook for reading the manuscript, and Mr. D, Stewart for photographic assistance.
Department of Anatomy, University of Western Australia, Nedlands, W.A. 6009 (Australia)
D. G. JONES
Brain Research, 29 (1971) 378-382
382
SHORT COMMUNICATIONS
1 COTTRELL, G. A., Separation and properties of subcellular particles associated with 5-hydroxytryptamine, with acetylcholine and with an unidentified cardio-excitatory substance from Mercenaria nervous tissue, Comp. Biochem. Physiol., 17 (1966) 891-907. 2 DE ROBERTIS, E., RODRIGUEZ DE LORES ARNAIZ, G., SALGANICOFF,L., PEELEGRINO DE IRALDI, A., AND ZIEHER, L. M., Isolation of synaptic vesicles and structural organization of the acetylcholine system within brain nerve endings, J. Neurochem., l0 (1963) 225-235. 3 FLOREY, E., AND WINESDOREER, J., Cholinergic nerve endings in octopus brain, J. Neurochem., 15 (1968) 169-177. 4 HORNE, R. W., AND WHITTAKER,V. P., The use of the negative staining method for the electronmicroscopic study of subcellular particles from animal tissues, Z. Zellforsch., 58 (1962) 1-16. 5 JONES, D. G., An electron-microscope study of subcellular fractions of Octopus brain, J. Cell Sci., 2 (1967) 573-586. 6 JONES, O. G., The fine structure of the synaptic membrane adhesions on octopus synaptosomes, Z. Zellforsch., 88 (1968) 457469. 7 JONES, D. G., A further contribution to the study of the contact region of Octopus synaptosomes, Z. Zellforsch., 103 (1970) 48-60. 8 MUSCATELLO, U., AND HORNE, R. W., Effect of the tonicity of some negative-staining solutions on the elementary structure of membrane-bounded systems, J. Ultrastruct. Res., 25 (1968) 73-83. 9 SHERIDAN,M. N., WHITTAKER, V. P., AND ISRAEL,M., The subcellular fractionation of the electric organ of Torpedo, Z. Zellforsch., 74 (1966) 291-307. 10 WHITTAKER, V. P., MICHAELSON, I. A., AND KIRKLAND, R. J. A., The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'), Biochem. J., 90 (I 964) 293-303. 11 WHITTAKER, V. P., AND SHERIDAN, M. N., The morphology and acetylcholine content of isolated cerebral cortical synaptic vesicles, J. Neurochem., 12 (•965) 363-372.
(Accepted April 2nd, 1971)
Brain Research, 29 (1971) 378-382