Histochemistry58, 193-201 (1978)
Histochemistry 9 by Springer-Verlag 1978
Morphometric Analyses of the Electric Organ of Torpedo: The Influence of Different Fixative Modes on the Vesicle Diameter W. Naef, K. Munz, and P.G. Waser Institute of Pharmacology, University of Ztirich, Gloriastrage32, CH-8006 Zfirich, Switzerland
Summary. The influences of various fixatives on the vesicle size of the electric organ of Torpedo marmorate were investigated. Thin section and freeze etched preparations were examined under the electron microscope. In thin section increased vesicle diameters were observed compared with the freeze etched preparations. The same experiments in different torpedo fish led to significantly different vesicle sizes observed. Variations of the molarity, the pH and osmolarities result in particularly high differences in vesicle diameters. Using Karnovsky's method (1965) and a fixative consisting of 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, results in vesicle sizes comparable to those reported by other authors. Results obtained from freeze etched preparations are not comparable in general with results from thin section experiments with the same fixative.
Introduction Torpedo electric organs are of increasing research interest, in part because they represent a unique source from which large quantities of cholinergic nerve endings with synaptic vesicles can be obtained. Since the initial descriptions of synaptic vesicles (de Robertis and Bennett, 1955) and their hypothesized relationship to the secretory and electrophysiological phenomena associated with synaptic transmission (del Castillo and Katz, 1954), many scientists have attempted to determine the size of these vesicles in the electric organ of 7brpedo marmorata (Sheridan, 1965; Israel et al., 1970; Nickel and Potter, 1971; Naef and Waser, 1975; Zimmermann, 1975). The vesicle diameters were described by different workers to be in the range of 30-120 nm. Our own previous measurements (Naef and Waser, 1975) resulted in the diameters of about 65 nm. In the present paper we report morphometric measurements of synaptic vesicles of T. marmorata (Fig. 1). It is of particular interest to see if any influence
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Fig.
1. Typical synapse of the electric organ of Torpedo marmorata. Abbreviations used: sv, synaptic vesicles; se synaptic cleft;prs, presynaptic side;pos, postsynaptic side
of fixatives on sizes of the synaptic vesicles, especially with regard to the varying results in the cited literature is observed. Therefore we tested and measured diameters of various types of fixation on samples prepared by both thin sectioning or freeze etching. We tried to establish a better fixation mode suitable for further morphometric studies.
Materials and Methods Animals. Experiments were performed on T. marmorata supplied by air freight from the Station Biologique d'Arcachon, France. Most of the fish were females of 30-60 cm in length. They were kept in seawater at about 15 ~ C.
Fixatives Influences on Vesicles Diameter
195
Table 1 Nr. of Group
Mode of fixation
Fish 1 1 2 3
1.5% G A in caco 0.2 M pH 7.2 2.5% G A in caco 0.2 M pH 7.2 5.0% G A in caco 0.2 M p H 7.2
4 5 6 7 8 9 10
2.5% 2.5% 2.5% 2.5% 2.5% 2.5% 2.5%
GA GA GA GA GA GA GA
in in in in in in in
caco caco caco caco caco caco caco
0.2 0.2 0.2 0.1 0.2 0.3 0.4
M M M M M M M
pH pH pH pH pH pH pH
5.5 7.2 8.5 7.2 7.2 7.2 7.2
11 12 13 14
5.0% 5.0% 2.5% 2.5%
GA/4.0% F A in caco 0.1 M pH 7.2 G A in caco 0.2 M pH 7.2 G A in caco 0.2 M p H 7 . 2 + s u c r o s e G A in caco 0.2 M p H 7 . 2 + s u c r o s e
Fish 2 1 2 3
1.5% G A in caco 0.2 M p H 7.2 2.5% G A in caco 0.2 M p H 7.2 5.0% G A in caco 0.2 M p H 7.2
Abbreviations used: GA, glutaraldehyde; FA, formaldehyde; caco, cacodylate buffer
Histology. The fish were anaesthetized by placing them in seawater containing 0.04% ethyl m-aminobenzoate methane sulphonate (triacine methane sulphonate, MS-222 | Sandoz). The ultrastructural studies were carried out on small pieces (lmm 3) of the electric organ of T. marmorata. The epidermis covering the electric organ was removed and small organ pieces were rapidly fixed in the different fixatives (Table 1) for 90 rain at 4 ~ C. After a brief fixation (about 20 rain), some of the organ pieces of each fixative solution were taken out and prepared for freeze etching. They were soaked for 20 rain in 20% glycerol in Ringer's solution for cryoprotection and finally frozen in Freon cooled by liquid nitrogen. The remainder were prepared for ultrastructural studies on thin section by washing in the corresponding cacodylate buffer (Table 1) for several hours. Then these pieces were osmicated by immersion in 2% OsO4. After dehydration in ethanol and embedding in Araldite, silver gray thin sections were cut with a LKB ultra microtome. They were stained with uranyl acetate and lead citrate. The frozen tissue pieces were fractured, etched and shadowed with carbon and platinum to produce replicas, in a Balzers Freeze Etching Unit 360 M. Photography. Electron microscopy was done with a Philips 201 electron microscope calibrated with a carbon grating replica. All micrographs were taken at the same magnification and photographically enlarged. Morphometric analysis. The quantitative analysis of the resulting photographs was accomplished with a manual optical system for m o r p h o m e t r y linked to a desk computer (Olivetti P 602). The measurements of the size of vesicles were made with an electro-optical pencil and a contact grid plate. A b o u t 500 vesicles on 10 different photographs of two organ pieces were measured to form one group. The results of the various experimental groups were compared to one another with Student-t-tests (error probability of p < 0.05, significances are two-tailed).
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l
! / //
85
80 / f g,
/
75 /
Group
/
thin section ------freeze etching
J
/
I
I
I
1
2
3
Fig. 2. Vesicle diameter of synapses, fixed in glutaraldehyde of various concentrations in 0.2 M cacodylate buffer, pH 7.2 Group Concentration of fixative Vesicle ~ thin section (nm) Vesicle ~ freeze etching (nm)
1 1.5% 77.9 _+4.8 a 72.7 -+5.9
2 2.5% 79.6 • 5.2 ~ 76.9 • 5.9
3 5.0% 83.2 -+4.6 86.0 _+5.3
Values are mean _+S.E.M. "
Significant agreement (p > 0,05)
Results
Changes of Fixative Concentrations. The fixative c o n t a i n i n g 1.5 % g l u t a r a l d e h y d e ( G r o u p 1) led to a vesicle d i a m e t e r o f 77.9 n m + 4 . 8 nm. The vesicle d i a m e t e r , obtained in the experiment ( G r o u p 2) with 2.5% glutaraldehyde ( ~ 7 9 . 6 n m + 5.2 nm) showed no significant differences to the d a t a o f g r o u p 1 ( p > 0 . 0 5 ) . The s i t u a t i o n was s o m e w h a t different with 5.0% g l u t a r a l d e h y d e ( G r o u p 3). T h e vesicle size o f 83.2 n m + 4 . 6 n m is significantly different f r o m the sizes o b t a i n e d with all the o t h e r c o n c e n t r a t i o n s (p < 0.05) (Fig. 2). The values f r o m the freeze etched p r e p a r a t i o n s also increased f r o m the 1.5% g l u t a r a l d e h y d e c o n t a i n i n g fixative ( G r o u p 1) to the 5% g l u t a r a l d e h y d e c o n t a i n i n g fixative ( G r o u p 3). The vesicle sizes f o u n d u n d e r the v a r i o u s conditions are all significantly different ( p < 0 . 0 5 ) f r o m each other. In c o m p a r i s o n Table 2 Group
1
2
Concentration of fixative Vesicle ~ thin section (nm) Vesicle ~ freeze etching (nm)
1.5% 93.7_+4.3 75.6_+5.8
2.5% 5.0% 86.2_+4.5 90.9+4.7 b 89.5+_5.3a ~9.5-+5.4"'~
Values are mean+S.E.M. b
Significant agreement (p > 0.05) Significant agreement (p > 0.05)
3
Fixatives Influences on Vesicles Diameter
197
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75 Group
4
5
6
Fig. 3. Vesicle diameter of synapses, fixed in 2.5% glutaraldehyde in 0.2 M cacodylate buffer, with various pH Group pH Vesicle ~ thin section (nm) Vesicle ~ freeze etching (rim)
4 5.5 90.2 • 4.7 a, b 88.9 _+5.4 a
5 7.2 79.6 _+5.2 76.9 • 5.9
6 8.5 90.8 _+5.0 b 82.2 • 5.2
Values are mean • S.E.M.
a b
Significantagreement (p >0.05) Significantagreement (p > 0.05)
to the results Of thin section experiments, the freeze etched preparations show significantly (p< 0.05) lower vesicle diameters with the exception of the vesicle sizes obtained with the 5% glutaraldehyde containing fixative (Group 3). The same experiments were done with the electric organ of a second fish and led to the results in Table 2. There are significant differences (p 0.05). Only in the freeze etched preparations in the series with 1.5% glutaraldehyde in the fixation medium (Group 1) are the vesicle sizes in both experimental series (Fig. 1 and Table 2) equal. With the exception of 1.5% glutaraldehyde (Group 1) the vesicle sizes are larger in the experiments involving the second fish.
Changes of the pH. The pH of the fixative containing 2.5% glutaraldehyde and 0.2 M cacodylate buffer was varied in following manner: pH 5.5 (Group 4), pH 7.2 (Group 5), pH 8.5 (Group 6). The experiments were carried out in thin sections and in freeze etched preparations with the same fixative solution (Fig. 3).
198
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7
8
9
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10
Fig. 4. Vesicle diameter of synapses, fixed in 2.5 % glutaraldehyde in various molarities of cacodylate buffer, pH 7.2 Group Molarity Vesicle ~ ts (nm) Vesicle ~ fe (nm)
7 0.1 89.2 • 4.8 83.2 • 5.4
8 0.2 79.6 + 5.2 a 76.9 • 5.9 b
9 0.3 80.4 + 5.4 a 88.5 • 4.9
10 0.4 86.7 • 4,7 77.9 • 5.2 b
Values are mean • S.E.M.; ts, thin section; fe, freeze etching
" b
Significantagreement (p > 0.05) Significantagreement (p > 0.05)
In thin sections, fixation with pH 5.5 (Group 4) and pH 8.5 (Group 6) resulted in a notable increase of vesicle sizes without any significant differences between the diameters of the two experiments (p > 0.05) (Fig. 3). The fixation with a pH of 7.2 (Group 5) showed a significant decrease in vesicle diameter (p 0.05).
Fixatives Influences on Vesicles Diameter
199
85
|
80
/
~
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I -
-
-
-
-
-
75 Group
freeze etching
i
i
L
h
ll
12
13
14
Fig, 5. Vesiclediameter of synapses, fixed in gtutaratdehyde (exception Group 1l with glutaraldehyde/ formaldehydefixation medium and 0.1 M cacodylatebuffer) in 0.2 M cacodylatebuffer, pH 7.2 with various osmolarities Group Osmolarity (mosm) Vesicle ~ ts (nm) Vesicle ~ fe (nm)
11 735 79.9_+4.3 75.6+_6.6
12 795 83.2_+4.6 86.9+5.3 a
13 900 87.6_+4.7 b 86.3_+4.4 "'b
14 1150 77.6+_4.7 85.5+4.6 a
Values are mean_+ S.E.M. ; ts, thin section; fe, freeze etching
a b
Significantagreement (p > 0.05) Significantagreement (p > 0.05)
Changes of Osmolarity. To demonstrate the influence of osmolarity on the vesicle size in thin section experiments, fixatives with different osmolarities were used (Fig. 5). The groups with high osmolarities were adjusted with sucrose. These experiments showed an increase of vesicle sizes corresponding to the increase of osmolarity up to 900 mosm. In the highest sucrose concentration (1150 mosm, Group 14) the vesicle diameter drastically decreased to a level which was almost the same as with the sample of 735 mosm (Group 11). All the results obtained from these experiments were significantly different to one another (p 0.05). Only the sample of 735 mosm (Group 11) was significantly different from all others (p < 0.05). Between freeze etched and thin section samples agreemerits are seen in Group 13 (p>0.05). Discussion The influences of various procedures of fixation on the size of synaptic vesicles in the electric organ of torpedo is higher than expected. This concerns especially
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W. Naef et al.
the experiments with the lowest or highest fixative concentrations and pH. These conditions yielded sizes of vesicles larger than those described by different scientists (Israel et al., 1970; Nickel and Potter, 1971; Naef and Waser, 1975; Zimmermann, 1975). Although Israel et al. (1970) used a similarly high concentration (6.5% glutaraldehyde), they described a vesicle size of only 80 nm, while our results were 83 nm and 91 nm in the different fish. This discrepancy is a clear indication of the difficulties when comparing vesicle sizes from experiments in different fish. In our own experiments only in the freeze etched preparations do two experiments with the same fixation in different fish (Group 2, Fig. 1) show no significant differences (p > 0.05). In thin section preparations there was disagreement between the different results in the various fish. This effect may result from the preparation technique used for freeze etching. But it is to be remembered that there is certainly a natural variation between the torpedos and, in addition, it was not possible to determine the age of the experimental.fish or to obtain animals from the same population. Generally the values obtained from the freeze etched preparations are lower than those from the thin section experiments. An explanation for this phenomenon could be the short time of fixation before the organ pieces were frozen by liquid nitrogen. In this case the osmolarities and concentrations of the fixatives are not so important. The reason for the large differences between the thin section and freeze etched preparations observed in the experiments with different molarities, mainly in the 0.3 M and 0.4 M groups (Fig. 4) and in the experiment with different osmolarities (Fig. 5) is not clear. The same effects are also visible in the results of the experiments using the second fish preparations, especially in the first experimental group with 1.5% glutaraldehyde. There is also a significant difference observable in this case between the results from the thin section and freeze etching technique. Compared to the results (75 nm) of Zimmermann (1975) who used the same fixative (Karnovsky, 1965) as we did in group 11, we found larger values of vesicle sizes than they did. This lack of accord can be explained by the natural differences in the cell compartments of the electric organs, or perhaps by the use of different measurement techniques. Technical reasons, such as errors in individual measurements or small variations in the magnification-factor of the electron microscope or the enlarging camera may also lead to small variations in the vesicle sizes. The influence of certain parameters, especially the pH and the molarities cause deviations which allow us to eliminate unsuitable fixation solutions for morphometric experiments. It also allows us to find the fixation solutions producing results comparable with data in most of the cited papers (Israel et al., 1970; Nickel and Potter, 1971 ; Naef and Waser, 1975; Zimmermann, 1975). In this regard Karnovsky's (1965) method (Group 11) and a solution of 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2 (Group 2, 5, 8) are suitable for the fixation of the electric organ of torpedo, especially if supplemented with sucrose for reaching an osmolarity of 800 mosm [ = osmolarity of the electric organ of torpedo (Dunant et al., 1976)].
Fixatives Influences on Vesicles Diameter
201
Suitables Fixatives for Histological Experiments. Comparisons of the different vesicle diameters in the various experiments led to the finding that only in experiments involving the first fish was the fixation medium usually used in histological techniques (1.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, Group 1) in good agreement with published data both on thin section preparations (Israel et al., 1970; Zimmermann, 1975) and fi'eeze etched preparations (Nickel and Potter, 1971). The fixation medium (2.5% glutaraldehyde in 0.2 m cacodylate buffer pH 7.2 gave in all the experiments the lowest variations (Figs. 1, 5, Table 2) and the vesicle sizes resemble those published by other workers (Israel etal., 1970; Zimmermann, 1975). The other series exhibit scatter and reveal no regularities, indicating therefore that these conditions are unsuitable for histomorphometric analysis. Acknowledgements. This work was supported by a grant Nr. 3.086.76 of the Swiss National Foundation for Scientific Research. We are grateful to Miss. C. Toscano and Miss C. Maier for their technical assistance.
References del Castillo, J., Katz, B.: Quantal components of the endplate potential. J. PhysioI. 124, 560 (1954) Dunant, Y., Israel, M., Manaranche, R.: Loss of vesicular acetylcholine in the Torpedo electric organ on discharge against high external resistance. J. Neurochem. 27, 975-977 (1976) Israel, M.,Gautron, J., Lesbats, B. : Fractionnement de l'organ electrique de la torpille: localisation subcellulaire de l'acetylcholine. J. Neurochem. 17, 1441-1450 (1970) Karnovsky, M.J.: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Ceil Biol. 27, 137A-138A (1965) Naef, W., Waser, P.G.: Changes in cholinergic synapses of Torpedo under the influence of drugs. In: Cholinergic mechanisms, pp. 67-77. New York: Raven Press 1975 Nickel, E., Potter, L.T. : Synaptic vesicles in freeze etched electric tissue of Torpedo. Phil. Trans. Roy. Soc. Lond. B. 261, 383-385 (1971) de Robertis, E.D., Bennett, H.S.: Some features of the submicroscopic morphology of synapses in frog and earthworm. J. Biophysic. Biochem. Cytol. 1, 47-63 (1955) Sheridan, M.N. : The fine structure of the electric organ of T. marmorata. J. Cell Biol. 24, 139-141 (1965) Zimmermann, H. : Dynamics of synaptic transmission in the cholinergic synapses of the electric organ of Torpedo. In: Cholinergic mechanisms, pp. 169-176. New York: Raven Press, 1975
Received July 14, 1978
Histochemistry 9 by Springer-Verlag 1978
Morphometric Analyses of the Electric Organ of Torpedo: The Influence of Different Fixative Modes on the Vesicle Diameter W. Naef, K. Munz, and P.G. Waser Institute of Pharmacology, University of Ztirich, Gloriastrage32, CH-8006 Zfirich, Switzerland
Summary. The influences of various fixatives on the vesicle size of the electric organ of Torpedo marmorate were investigated. Thin section and freeze etched preparations were examined under the electron microscope. In thin section increased vesicle diameters were observed compared with the freeze etched preparations. The same experiments in different torpedo fish led to significantly different vesicle sizes observed. Variations of the molarity, the pH and osmolarities result in particularly high differences in vesicle diameters. Using Karnovsky's method (1965) and a fixative consisting of 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, results in vesicle sizes comparable to those reported by other authors. Results obtained from freeze etched preparations are not comparable in general with results from thin section experiments with the same fixative.
Introduction Torpedo electric organs are of increasing research interest, in part because they represent a unique source from which large quantities of cholinergic nerve endings with synaptic vesicles can be obtained. Since the initial descriptions of synaptic vesicles (de Robertis and Bennett, 1955) and their hypothesized relationship to the secretory and electrophysiological phenomena associated with synaptic transmission (del Castillo and Katz, 1954), many scientists have attempted to determine the size of these vesicles in the electric organ of 7brpedo marmorata (Sheridan, 1965; Israel et al., 1970; Nickel and Potter, 1971; Naef and Waser, 1975; Zimmermann, 1975). The vesicle diameters were described by different workers to be in the range of 30-120 nm. Our own previous measurements (Naef and Waser, 1975) resulted in the diameters of about 65 nm. In the present paper we report morphometric measurements of synaptic vesicles of T. marmorata (Fig. 1). It is of particular interest to see if any influence
0301-5564/78/0058/0193/$01.80
194
W. Naef et al.
Fig.
1. Typical synapse of the electric organ of Torpedo marmorata. Abbreviations used: sv, synaptic vesicles; se synaptic cleft;prs, presynaptic side;pos, postsynaptic side
of fixatives on sizes of the synaptic vesicles, especially with regard to the varying results in the cited literature is observed. Therefore we tested and measured diameters of various types of fixation on samples prepared by both thin sectioning or freeze etching. We tried to establish a better fixation mode suitable for further morphometric studies.
Materials and Methods Animals. Experiments were performed on T. marmorata supplied by air freight from the Station Biologique d'Arcachon, France. Most of the fish were females of 30-60 cm in length. They were kept in seawater at about 15 ~ C.
Fixatives Influences on Vesicles Diameter
195
Table 1 Nr. of Group
Mode of fixation
Fish 1 1 2 3
1.5% G A in caco 0.2 M pH 7.2 2.5% G A in caco 0.2 M pH 7.2 5.0% G A in caco 0.2 M p H 7.2
4 5 6 7 8 9 10
2.5% 2.5% 2.5% 2.5% 2.5% 2.5% 2.5%
GA GA GA GA GA GA GA
in in in in in in in
caco caco caco caco caco caco caco
0.2 0.2 0.2 0.1 0.2 0.3 0.4
M M M M M M M
pH pH pH pH pH pH pH
5.5 7.2 8.5 7.2 7.2 7.2 7.2
11 12 13 14
5.0% 5.0% 2.5% 2.5%
GA/4.0% F A in caco 0.1 M pH 7.2 G A in caco 0.2 M pH 7.2 G A in caco 0.2 M p H 7 . 2 + s u c r o s e G A in caco 0.2 M p H 7 . 2 + s u c r o s e
Fish 2 1 2 3
1.5% G A in caco 0.2 M p H 7.2 2.5% G A in caco 0.2 M p H 7.2 5.0% G A in caco 0.2 M p H 7.2
Abbreviations used: GA, glutaraldehyde; FA, formaldehyde; caco, cacodylate buffer
Histology. The fish were anaesthetized by placing them in seawater containing 0.04% ethyl m-aminobenzoate methane sulphonate (triacine methane sulphonate, MS-222 | Sandoz). The ultrastructural studies were carried out on small pieces (lmm 3) of the electric organ of T. marmorata. The epidermis covering the electric organ was removed and small organ pieces were rapidly fixed in the different fixatives (Table 1) for 90 rain at 4 ~ C. After a brief fixation (about 20 rain), some of the organ pieces of each fixative solution were taken out and prepared for freeze etching. They were soaked for 20 rain in 20% glycerol in Ringer's solution for cryoprotection and finally frozen in Freon cooled by liquid nitrogen. The remainder were prepared for ultrastructural studies on thin section by washing in the corresponding cacodylate buffer (Table 1) for several hours. Then these pieces were osmicated by immersion in 2% OsO4. After dehydration in ethanol and embedding in Araldite, silver gray thin sections were cut with a LKB ultra microtome. They were stained with uranyl acetate and lead citrate. The frozen tissue pieces were fractured, etched and shadowed with carbon and platinum to produce replicas, in a Balzers Freeze Etching Unit 360 M. Photography. Electron microscopy was done with a Philips 201 electron microscope calibrated with a carbon grating replica. All micrographs were taken at the same magnification and photographically enlarged. Morphometric analysis. The quantitative analysis of the resulting photographs was accomplished with a manual optical system for m o r p h o m e t r y linked to a desk computer (Olivetti P 602). The measurements of the size of vesicles were made with an electro-optical pencil and a contact grid plate. A b o u t 500 vesicles on 10 different photographs of two organ pieces were measured to form one group. The results of the various experimental groups were compared to one another with Student-t-tests (error probability of p < 0.05, significances are two-tailed).
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l
! / //
85
80 / f g,
/
75 /
Group
/
thin section ------freeze etching
J
/
I
I
I
1
2
3
Fig. 2. Vesicle diameter of synapses, fixed in glutaraldehyde of various concentrations in 0.2 M cacodylate buffer, pH 7.2 Group Concentration of fixative Vesicle ~ thin section (nm) Vesicle ~ freeze etching (nm)
1 1.5% 77.9 _+4.8 a 72.7 -+5.9
2 2.5% 79.6 • 5.2 ~ 76.9 • 5.9
3 5.0% 83.2 -+4.6 86.0 _+5.3
Values are mean _+S.E.M. "
Significant agreement (p > 0,05)
Results
Changes of Fixative Concentrations. The fixative c o n t a i n i n g 1.5 % g l u t a r a l d e h y d e ( G r o u p 1) led to a vesicle d i a m e t e r o f 77.9 n m + 4 . 8 nm. The vesicle d i a m e t e r , obtained in the experiment ( G r o u p 2) with 2.5% glutaraldehyde ( ~ 7 9 . 6 n m + 5.2 nm) showed no significant differences to the d a t a o f g r o u p 1 ( p > 0 . 0 5 ) . The s i t u a t i o n was s o m e w h a t different with 5.0% g l u t a r a l d e h y d e ( G r o u p 3). T h e vesicle size o f 83.2 n m + 4 . 6 n m is significantly different f r o m the sizes o b t a i n e d with all the o t h e r c o n c e n t r a t i o n s (p < 0.05) (Fig. 2). The values f r o m the freeze etched p r e p a r a t i o n s also increased f r o m the 1.5% g l u t a r a l d e h y d e c o n t a i n i n g fixative ( G r o u p 1) to the 5% g l u t a r a l d e h y d e c o n t a i n i n g fixative ( G r o u p 3). The vesicle sizes f o u n d u n d e r the v a r i o u s conditions are all significantly different ( p < 0 . 0 5 ) f r o m each other. In c o m p a r i s o n Table 2 Group
1
2
Concentration of fixative Vesicle ~ thin section (nm) Vesicle ~ freeze etching (nm)
1.5% 93.7_+4.3 75.6_+5.8
2.5% 5.0% 86.2_+4.5 90.9+4.7 b 89.5+_5.3a ~9.5-+5.4"'~
Values are mean+S.E.M. b
Significant agreement (p > 0.05) Significant agreement (p > 0.05)
3
Fixatives Influences on Vesicles Diameter
197
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85
~ 80
\
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/
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75 Group
4
5
6
Fig. 3. Vesicle diameter of synapses, fixed in 2.5% glutaraldehyde in 0.2 M cacodylate buffer, with various pH Group pH Vesicle ~ thin section (nm) Vesicle ~ freeze etching (rim)
4 5.5 90.2 • 4.7 a, b 88.9 _+5.4 a
5 7.2 79.6 _+5.2 76.9 • 5.9
6 8.5 90.8 _+5.0 b 82.2 • 5.2
Values are mean • S.E.M.
a b
Significantagreement (p >0.05) Significantagreement (p > 0.05)
to the results Of thin section experiments, the freeze etched preparations show significantly (p< 0.05) lower vesicle diameters with the exception of the vesicle sizes obtained with the 5% glutaraldehyde containing fixative (Group 3). The same experiments were done with the electric organ of a second fish and led to the results in Table 2. There are significant differences (p 0.05). Only in the freeze etched preparations in the series with 1.5% glutaraldehyde in the fixation medium (Group 1) are the vesicle sizes in both experimental series (Fig. 1 and Table 2) equal. With the exception of 1.5% glutaraldehyde (Group 1) the vesicle sizes are larger in the experiments involving the second fish.
Changes of the pH. The pH of the fixative containing 2.5% glutaraldehyde and 0.2 M cacodylate buffer was varied in following manner: pH 5.5 (Group 4), pH 7.2 (Group 5), pH 8.5 (Group 6). The experiments were carried out in thin sections and in freeze etched preparations with the same fixative solution (Fig. 3).
198
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/
7
8
9
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freeze
etching
10
Fig. 4. Vesicle diameter of synapses, fixed in 2.5 % glutaraldehyde in various molarities of cacodylate buffer, pH 7.2 Group Molarity Vesicle ~ ts (nm) Vesicle ~ fe (nm)
7 0.1 89.2 • 4.8 83.2 • 5.4
8 0.2 79.6 + 5.2 a 76.9 • 5.9 b
9 0.3 80.4 + 5.4 a 88.5 • 4.9
10 0.4 86.7 • 4,7 77.9 • 5.2 b
Values are mean • S.E.M.; ts, thin section; fe, freeze etching
" b
Significantagreement (p > 0.05) Significantagreement (p > 0.05)
In thin sections, fixation with pH 5.5 (Group 4) and pH 8.5 (Group 6) resulted in a notable increase of vesicle sizes without any significant differences between the diameters of the two experiments (p > 0.05) (Fig. 3). The fixation with a pH of 7.2 (Group 5) showed a significant decrease in vesicle diameter (p 0.05).
Fixatives Influences on Vesicles Diameter
199
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|
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/
~
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I -
-
-
-
-
-
75 Group
freeze etching
i
i
L
h
ll
12
13
14
Fig, 5. Vesiclediameter of synapses, fixed in gtutaratdehyde (exception Group 1l with glutaraldehyde/ formaldehydefixation medium and 0.1 M cacodylatebuffer) in 0.2 M cacodylatebuffer, pH 7.2 with various osmolarities Group Osmolarity (mosm) Vesicle ~ ts (nm) Vesicle ~ fe (nm)
11 735 79.9_+4.3 75.6+_6.6
12 795 83.2_+4.6 86.9+5.3 a
13 900 87.6_+4.7 b 86.3_+4.4 "'b
14 1150 77.6+_4.7 85.5+4.6 a
Values are mean_+ S.E.M. ; ts, thin section; fe, freeze etching
a b
Significantagreement (p > 0.05) Significantagreement (p > 0.05)
Changes of Osmolarity. To demonstrate the influence of osmolarity on the vesicle size in thin section experiments, fixatives with different osmolarities were used (Fig. 5). The groups with high osmolarities were adjusted with sucrose. These experiments showed an increase of vesicle sizes corresponding to the increase of osmolarity up to 900 mosm. In the highest sucrose concentration (1150 mosm, Group 14) the vesicle diameter drastically decreased to a level which was almost the same as with the sample of 735 mosm (Group 11). All the results obtained from these experiments were significantly different to one another (p 0.05). Only the sample of 735 mosm (Group 11) was significantly different from all others (p < 0.05). Between freeze etched and thin section samples agreemerits are seen in Group 13 (p>0.05). Discussion The influences of various procedures of fixation on the size of synaptic vesicles in the electric organ of torpedo is higher than expected. This concerns especially
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the experiments with the lowest or highest fixative concentrations and pH. These conditions yielded sizes of vesicles larger than those described by different scientists (Israel et al., 1970; Nickel and Potter, 1971; Naef and Waser, 1975; Zimmermann, 1975). Although Israel et al. (1970) used a similarly high concentration (6.5% glutaraldehyde), they described a vesicle size of only 80 nm, while our results were 83 nm and 91 nm in the different fish. This discrepancy is a clear indication of the difficulties when comparing vesicle sizes from experiments in different fish. In our own experiments only in the freeze etched preparations do two experiments with the same fixation in different fish (Group 2, Fig. 1) show no significant differences (p > 0.05). In thin section preparations there was disagreement between the different results in the various fish. This effect may result from the preparation technique used for freeze etching. But it is to be remembered that there is certainly a natural variation between the torpedos and, in addition, it was not possible to determine the age of the experimental.fish or to obtain animals from the same population. Generally the values obtained from the freeze etched preparations are lower than those from the thin section experiments. An explanation for this phenomenon could be the short time of fixation before the organ pieces were frozen by liquid nitrogen. In this case the osmolarities and concentrations of the fixatives are not so important. The reason for the large differences between the thin section and freeze etched preparations observed in the experiments with different molarities, mainly in the 0.3 M and 0.4 M groups (Fig. 4) and in the experiment with different osmolarities (Fig. 5) is not clear. The same effects are also visible in the results of the experiments using the second fish preparations, especially in the first experimental group with 1.5% glutaraldehyde. There is also a significant difference observable in this case between the results from the thin section and freeze etching technique. Compared to the results (75 nm) of Zimmermann (1975) who used the same fixative (Karnovsky, 1965) as we did in group 11, we found larger values of vesicle sizes than they did. This lack of accord can be explained by the natural differences in the cell compartments of the electric organs, or perhaps by the use of different measurement techniques. Technical reasons, such as errors in individual measurements or small variations in the magnification-factor of the electron microscope or the enlarging camera may also lead to small variations in the vesicle sizes. The influence of certain parameters, especially the pH and the molarities cause deviations which allow us to eliminate unsuitable fixation solutions for morphometric experiments. It also allows us to find the fixation solutions producing results comparable with data in most of the cited papers (Israel et al., 1970; Nickel and Potter, 1971 ; Naef and Waser, 1975; Zimmermann, 1975). In this regard Karnovsky's (1965) method (Group 11) and a solution of 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2 (Group 2, 5, 8) are suitable for the fixation of the electric organ of torpedo, especially if supplemented with sucrose for reaching an osmolarity of 800 mosm [ = osmolarity of the electric organ of torpedo (Dunant et al., 1976)].
Fixatives Influences on Vesicles Diameter
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Suitables Fixatives for Histological Experiments. Comparisons of the different vesicle diameters in the various experiments led to the finding that only in experiments involving the first fish was the fixation medium usually used in histological techniques (1.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, Group 1) in good agreement with published data both on thin section preparations (Israel et al., 1970; Zimmermann, 1975) and fi'eeze etched preparations (Nickel and Potter, 1971). The fixation medium (2.5% glutaraldehyde in 0.2 m cacodylate buffer pH 7.2 gave in all the experiments the lowest variations (Figs. 1, 5, Table 2) and the vesicle sizes resemble those published by other workers (Israel etal., 1970; Zimmermann, 1975). The other series exhibit scatter and reveal no regularities, indicating therefore that these conditions are unsuitable for histomorphometric analysis. Acknowledgements. This work was supported by a grant Nr. 3.086.76 of the Swiss National Foundation for Scientific Research. We are grateful to Miss. C. Toscano and Miss C. Maier for their technical assistance.
References del Castillo, J., Katz, B.: Quantal components of the endplate potential. J. PhysioI. 124, 560 (1954) Dunant, Y., Israel, M., Manaranche, R.: Loss of vesicular acetylcholine in the Torpedo electric organ on discharge against high external resistance. J. Neurochem. 27, 975-977 (1976) Israel, M.,Gautron, J., Lesbats, B. : Fractionnement de l'organ electrique de la torpille: localisation subcellulaire de l'acetylcholine. J. Neurochem. 17, 1441-1450 (1970) Karnovsky, M.J.: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Ceil Biol. 27, 137A-138A (1965) Naef, W., Waser, P.G.: Changes in cholinergic synapses of Torpedo under the influence of drugs. In: Cholinergic mechanisms, pp. 67-77. New York: Raven Press 1975 Nickel, E., Potter, L.T. : Synaptic vesicles in freeze etched electric tissue of Torpedo. Phil. Trans. Roy. Soc. Lond. B. 261, 383-385 (1971) de Robertis, E.D., Bennett, H.S.: Some features of the submicroscopic morphology of synapses in frog and earthworm. J. Biophysic. Biochem. Cytol. 1, 47-63 (1955) Sheridan, M.N. : The fine structure of the electric organ of T. marmorata. J. Cell Biol. 24, 139-141 (1965) Zimmermann, H. : Dynamics of synaptic transmission in the cholinergic synapses of the electric organ of Torpedo. In: Cholinergic mechanisms, pp. 169-176. New York: Raven Press, 1975
Received July 14, 1978
