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Isolation of synaptic complexes from frozen and thawed brain tissue affected by multiple sclerosis
EXPERIMENTAL
Isolation
S.
48, 231-240
NEUROLOGY
(1975)
of Synaptic Complexes from Frozen and Thawed Tissue Affected by Multiple Sclerosis U.
KORNGUTH,
J. Departnke?kfs U&persity
of
VARAKIS,
JUHL,
T. D.
G.
JOHNSON, FUCCILLO,
SCOTT,
AND
J.
L. SEVER
Brain
KNOBELOCH, 1
Neurology,
of bzlisconsin National
Physiological Chemistry, Akkatonky, akad Pathology; Medical CePkter 53706, a& the Ixfcctious Discase Branch, Iastitute of Neurological D&use and Stroke, NIH, Bethesda, Maryland 20014 Receizfed
Febrzkary
20, 1975
The report compares the characteristics of isolated synaptic complexes (axon terminals retaining adhesion to the postsynaptic membrane) from frozen and unfrozen human brains (three from multiple sclerosis patients). All brains were obtained postmortem. The buoyant density in CsCl sucro’se density gradients of the synaptic complexes from frozen or fresh brain were identical (p 1.18-1.19) to each other and to the density of synaptic complexes from other humans and other species. Isolated synaptic complexes were readily recognizable by electron microscopy after freezing and thawing of the tissue. The membrane protein composition and buoyant density of the isolated synapses from frozen-thawed brains of patients who died of glomerular nephritis or multiple sclerosis were identical to each other in these parameters and to synapses from unfrozen brains. These fiindings indicate that frozen human brain may be used for several studies involving isolated synapses.
INTRODUCTION Intact synaptic junctions have been successfully isolated from fresh brain of a variety of species (1, 5-7, 11, 13, 14, 26) including man (23). When CsCl-sucrose gradients were used for isolation of synaptic endings, the equilibrium buoyant density of these intercellular organelles was 1.1751.190 whether the tissue used was from guinea pig (ll), swine (12-14)) 1 This work was supported by contract No. NS4-2308 and Grant NS 5631 from NINDS and by a gift from Dr. and Mrs. Leonard Weiss, Madison, Wisconsin. The authors express their appreciation to Dr. G. ZuRhein of the Department of Pathology, University of Wisconsin Medical Center for dissection of the tissue and advice in preparation of the manuscript. 231 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form re.wved
232
KORNGUTH
ET
AL.
monkey (14) or man (15, 23). The buoyant density of the synaptic endings was not affected either by the stage of ontogeny of the animal (12, 15, 23) or by the existence of various neuropathological states in man including hamartoma, Menke’s Kinky Hair syndrome, dementia, and various tumors (15, 23). The only disease observed to cause an alteration in the buoyant density of synapses was Tay-Sachs disease (15). In all of these above studies, unfrozen tissue was used (obtained within 20 hr postmortem). For the investigator interested in studying brains from diseased subjects, it is not always possible to obtain fresh tissue. For example, although fresh brain tissue from patients who died with multiple sclerosis is not readily available, there is an excellent bank of frozen brain specimens (collected by Dr. W. Tourtellotte). The buoyant density of synapses from multiple sclerosis tissue was of particular interest because of the reports that serum from multiple sclerosis patients affects synaptic transmission (3, 4) and the further observation (17) that unusual tubules (18 nm in diameter) were present in postsynaptic regions of six such patients. If these tubules were indeed viruslike structures containing mucleic acid, the buoyant density of the synaptic material might be increased. In this report it will be shown that well preserved synaptic complexes can be readily recovered from frozen brain tissues and that the buoyant density of synapses is 1.18-1.19 which is typical of synapses from other human brains (15, 23). Synapses from frozen multiple sclerosis samples also had normal buoyant densities. MATERIALS
AND
METHODS
To evaluate the effect of freezing and thawing on the isolation and buoyant density of synapses, sections of the frontal and temporal lobes were obtained three hours postmortem from a 13 year old female who died with autoimmune glomerular nephritis (Goodpasture syndrome). A portion of each region was processed immediately for isolation of synapses, and a second portion was frozen at -80 C for 24 hr prior to study. Gross and microscopic examination of this brain revealed no distinct cortical lesions. Frozen samples of brains from an individual who died with multiple sclerosis was obtained from the West Allis Memorial Hospital (with the cooperation of Ms. Lucille Uhlig, Multiple Sclerosis Society of Milwaukee) and two from the multiple sclerosis brain collection of Dr. W. Tourtellotte (University of California at Los Angeles), The specimens had been frozen for at least 1 year prior to study. Tissue of frontal and occipital lobes was used for isolation of synapses. After thawing at room temperature (22 C) for 30-60 min, cerebral white matter was dissected by scalpel away from the gray matter and the pial
SYNAPTIC
COMPLEXES
233
membrane was stripped off. Only the gray matter itself was processed for isolation of synapses. The white matter from all samples was used in other experiments not described in this report. Small portions of each brain were fixed intact to permit comparison of subcellular elements with structures existing in ritzy. The pieces (+ 27 mm”) were placed in cold cacodylate-buffered (0.1 ~1, pH 7.2) glutaraldehyde (2.5%) for 30-45 min. The tissues were rinsed for 15 min in the same buffer without glutaraldehyde, postfixed for 1 hr with 1% 0~04 in the same cacodylate buffer, and processed for embedding in Araldite. Isolation of Synaptic Complexes. The dissected gray regions from the cases with autoimmune glomerular nephritis and multiple sclerosis were placed immediately in sucrose solution A (0.32 M sucrose, 0.001 M MgClz and 0.0004 M phosphate buffer, pH 7.0). The tissue was homogenized in 5 vol of sucrose solution A with a Dounce tissue grinder. The homogenate was centrifuged at 1lOOg for 20 min and the resultant supernatant was decanted and centrifuged at 11,OOOg for 40 min. The ll,OOOg pellet was suspended in sucrose solution A (for each gram of original gray matter used there were 8 ml of suspending sucrose solution A). An aliquot of this suspension (8.32 ml) was mixed with 1.68 ml saturated CsCl (previously adjusted to pH 7.0) and then placed in polyallomer tubes for use in the SW 41 Beckman rotor. Density gradient beads (Beckman Co.) for the range of 1.10 and 1.20 g/cm3 were added to each tube. The density gradient was generated during a 66 hr centrifugation at 36,000 rpm in the LS-50 ultracentrifuge with the SW 41 rotor. After centrifugation, each visible band was removed by puncturing the polyallomer tube with a needle (20 gauge) and withdrawing the band into a syringe. The bands were diluted with 2 vol of sucrose A and centrifuged at 100,OOOg for 1 hr in the 50 rotor. Portions of the pellets were processed for electron microscopic analysis and the remainder used for biochemical analysis. Electvorz Il~icvoscopy. Pellets were fixed for 1 hr in 1% 0~04 buffered with 0.1 M cacodylate (pH 7.2) and processed for embedding in Araldite. Thin sections were contrasted with uranyl acetate and lead citrate. They were examined and photographed either in an AEI 801 or Philips EM-200 electron microscope. Electrophoresis of Proteins. The fractions recovered from the CsCl-sucrose gradient were lysed by suspension in 10 vol of distilled water. The lysate was centrifuged at 10,OOOg for 40 min and this procedure repeated two additional times. The final insoluble membrane fractions were solubilized with 1% sodium dodecyl sulfate. The resultant solutions were subjected to electrophoresis by the method of Weber and Osborn (25) for 18 hr on 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate and 0.1 M phosphate buffer (pH 7.0) using a constant current of
234
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ET
AL.
3 ma per gel. Gels were then stained with Coomassie blue for 1 hr and destained in 5% methanol and 9.2% acetic acid in water. Gels were photographed and stored in an aqueous solution of 7.5% acetic acid. RESULTS Isolation of tlze Synaptic Complexes. Each of the fractions from unfrozen gray matter and frozen gray matter were similar with one exception. The 1lOOg pellet obtained from the homogenate of fresh ,tissue had three layers : (i) a discrete pink zone at the tube bottom which contained primarily nuclei ; (ii) a loose white layer above the pink ; (iii) a white filmlike layer on top. The pink nuclear zone had sufficient cohesiveness to permit its isolation from the above two layers. The 1lOOg pellet recovered from the frozen tissue had no discrete nuclear zone although the lower portion of the pellet had a pink hue. Although intact nuclei could be readily identified by phase microscopy of the pellet from frozen brain, they were mixed in with other cell components to a much greater extent than in the pink zone of unfrozen brain pellets. After centrifugation of the crude mitochondrial fraction (11,OOOg pellet) in the CsCl-sucrose gradient, the banding pattern of material from fresh and frozen brain samples appeared very similar (Fig. la, b). There is a band (A) at density 1.10 which contains myelin as determined by ultrastructural study. A second band (p 1.14-1.16) (B) has the majority of particulate material and a third band (C) 1.18-1.19 is apparent above the 1.2 density marker bead. A fourth granular band (D) appears below the 1.2 marker. The banding pattern in the CsCl sucrose gradient of the material from the multiple sclerosis brains (Fig. lc) resembled that of the patient with autoimmune glomerular nephritis with two exceptions. Zone B from the multiple sclerosis tissue was much reduced in amount and was distributed over a larger density region (from 1.13 to 1.17) compared to a distinct band B from the autoimmune glomerular nephritis brain. Band D from the multiple sclerosis material was at 1.20-1.21 and was more compact than band D from the autoimmune glomerular nephritis disease. Band C in both cases ranged between 1.18-l. 19 (this band contained intact synaptic endings and is described below), The tube c was drawn from the fractionation of one of the multiple sclerosis patients. Patterns from the other two multiple sclerosis patients were identical to this illustrated pattern. Electron Microscopy. Synapses were readily identified in the frozenthawed tissue prior to homogenization (Fig. 2 insets) and after homogenization and centrifugation (Fig. Za, b). The majority of all synaptic complexes recovered from the CsCl sucrose gradient were localized in band C, and the major components present in band C were synaptic end-
S17NAPTIC
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b
FIG. 1. Distribution of membranes from unfrozen control, frozen control and frozen multiple sclerosis tissue in the cesium chloride density gradient. The fraction applied to the gradient was from the 11,OOOg pellet resuspended in sucrose A. Prior to centrifugation 8.32 ml of the suspension was mixed with 1.68 ml of saturated CsCl solution and the mixture was centrifuged for 66 hr. The filled circle at the region of band A represents the density gradient bead (p 1.10) and the filled circle by band C or D represents the density gradient bead (p 1.20). Tube a is from the unfrozen control brain, tube b is from the frozen and thawed control brain; tube c is from the multiple sclerosis case. Band C contained the isolated synaptic complexes ; electron microscopic photographs of these synaptic complexes are shown below the drawing of each tube. The presynaptic region is desi,gnated s. (X 28,050).
236
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ET
AL.
FIG. 2. Electron micrographs of membranes in band C recovered after fractionation of the brains obtained from patients who died with multiple sclerosis. Figure Za is from one patient and Fig. 2b is a sample from a second patient. Both tissues were obtained in a frozen condition prior to study. Several isolated synaptic endings are designated S. Magnification is 30,400 for a and 27,600 for b. The insets are photographs
SYNAPTIC
COMPLEXES
237
ings (Fig. 2). After freezing and thawing of human brain tissue prior to homogenization the presynaptic and postsynaptic elements were readily identifiable (Fig. la, b, c) and retained their adhesion. Of particular interest was the finding that intact synapses could be isolated from multiple sclerosis brains (Fig. 2) that had been frozen at -80 C for extended periods (longer than 1 year) and that these synaptic complexes had a buoyant density typical of synapses prepared from unfrozen human brains ( 1.175-l. 19). Membrane-enclosed glycogen granules were observed in band C as previously reported (11) for guinea pig brains fractionated in CsCl density gradients. Narang and Field (17) reported tubular arrays in postsynaptic regions of multiple sclerosis brains and suggested that these arrays may be viruses or neurotubules. In the present study, clear tubular arrays were not seen in any fraction, This may reflect either absence of 18 nm tubular structures in the brains examined or destruction of their ultrastructure by the freeze-thaw procedure similar to that seen after freezing of measles virus (24). Band A from the autoimmune glomerular nephritis patient contained primarily myelin fragments with the usual appearance. Band A from the multiple sclerosis brains however contained lysosomes and degenerative myelin in addition to myelin. Band B contained many membraneous fragments of undefined origin and fragments of smooth endoplasmic reticulum. Band D contained free mitochondria, filamentous material and chromatinlike fibrils. Chromatin was most likely released from the nuclei ruptured by the freezing process (evidenced by loss of the discrete pink zone described above). Electrophoretic Analysis of Synaptic Proteins. The protein components of the synaptic membranes recovered from the CsCl-sucrose gradients are shown in Fig. 3. Synapses from multiple sclerosis brains and autoimmune glomerular nephritis brains had similar protein patterns on polyacrylamide sodium dodecyl sulfate gels. The major protein species have molecular weights 92,000, 53,000, and 43,000. The protein analysis on polyacrylamide gels reveals a similar molecular weight distribution of proteins in synapses from patients having either multiple sclerosis or autoimmune glomerular nephritis. This protein pattern is similar to that of synapses from other human brains ( 15,22). DISCUSSION This report shows that the process of freezing does not modify the buoyant density of isolated synaptic complexes and that these intercellular of synapses present in the frozen-thawed brains fixed prior to homogenization and centrifugation. Each inset is of the brain sample used for isolation shown in Fig. 2a and b. Inset a is 28,178 X and b is 28,730 X.
238
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ET
AL.
patterns of proteins in isolated synaptic complexes from the FIG. 3. Electrophoretic control brain, the multiple sclerosis brain and swine brain. The membranes in fraction C from the CsCl gradients were solubilized with 1% sodium dodecyl sulfate and subjected to electrophoresis for 18 hr on 10% polyacrylamide gels (0.1 M phosphate buffer, pH 7.0) containing 0.1% sodium dodecyl sulfate. The current was 3 ma per gel with the cathode at the top and anode at the bottom. Gels were stained with Coomassie blue for 1 hr and destained in methanol: acetic acid. Gel a is of the swine proteins, b of unfrozen controls, c of frozen controls and d of multiple sclerosis fractions. The protein pattern from each gel is similar to that of the other gels.
organelles can be recognized in the electron microscope. Additionally, synapses from brain tissue affected by a prolonged condition of multiple sclerosis have buoyant densities (1.18-1.19) in CsCl and an ultrastructure and protein pattern that is identical to those of synapses from other species (11, 14) and other human brains except from Tay-Sachs disease (15,22, 23). The observation that frozen-thawed brain can be used for certain ultrastructural and biochemical investigations of synaptic elements should facilitate research efforts with respect to neural disorders. The researcher can use brain samples kept in a frozen state for several years or can receive frozen
samples
from
distant
laboratories.
The findings that synapsesfrom multiple sclerosis brains have the same buoyant density as synapsesfrom other brains is interesting becauseof the postulated role of slow viruses in multiple sclerosis (2, S-10, 16-21). Only
SYNAPTIC
CO&IPLESES
239
one of these reports (17) suggests that viruses may be localized near synapses whereas the others involve the region adjacent to the plaque. The subcellular components containing aggregates of viruses may be expected to have an increased buoyant density because of the high density of nucleic acid (161.7). None of the fractions from gray matter (including synapses) of the multiple sclerosis tissues examined have an increased buoyant density, and on this basis it appears that synaptic complexes isolated from multiple sclerosis brain do not contain high concentration of nucleocapsid. In summary, we conclude that multiple sclerosis does not cause the appearance of membrane-associated synaptic proteins having unusual molecular weight distributions and that the freezing process does not markedly affect the buoyant density or the protein composition of synaptic components. REFERENCES 1. ABDEL-LATIF, A. A. 1961. A simple from rat brain. Bioclzinr. Biophys. 2. ADAMS, J. M., and D. T. IE*IAGAWA.
method
for
isolation of nerve ending 4033406. 1962. Measles antibodies in multiple
particles
Acta 121:
sclerosis.
Proc. Sot. Exp. Biol. Med. 111: 562-566. 3. BORNSTEIN,
M. B., and S. M. CRAIN. 1965. Functional studies of brain tissues as related to “demyelinative disorders.” Science 148 : 1242-1244. 4. CERF, J. A., and G. CARELS. 1966. Multiple sclerosis: serum factor producing reversible alterations in bioelectric responses. Science 152 : 1066-1068. 5. COTMAN, C. W. 1974. Isolation of synaptosomal and synaptic plasma membrane fractions. In. “Methods in enzymology,” Vol. 31. S. Fleischer and L. Packer [Eds.]. Academic Press, New York. 6. DAY, E. D., P. N. MCMILMN, D. D. MICKEY, and S. H. APPEL. 1971. Zonal centrifuge profiles of rat brain homogenates: Instability in sucrose, stability in iso-osmotic ficoll-sucrose. Anal. Biockem 39: D-45. 7. DEROBERTIS, E. D. P. 1964. “Histophysiology of Synapses and Neurosecretion.” Academic Press, New York. 8. DUBOIS-DALCQ, M., G. SCHUMACHER, and J. L. SEVER. 1973. Acute multiple sclerosis: Electron microscopic evidence for and against a viral agent in the plaques. Lancet 2 : 1408-1411. 9. FIELD, E. J., S. COWSHALL, H. R. NARANG, and T. M. BELL. 1972. Viruses in multiple sclerosis? Lancet 2 : 280-281. 10. FUCCILLO, D. A., J. KURENT, and J. L. SEVER. 1974. Slow virus diseases. Am
Rev. Microhiol.
28 : 231-264.
11. KORNGUTH, S. E., J. W. ANDERSON, and G. SCOTT. 1969. Isolation of synaptic complexes in a cesium chloride density gradient: Electron microsopic and immunohistochemical studies. J. Nezfrochcnt. 16 : 1017-1024. 12. KORNGUTH, S., A. FLANGAS, R. GEISON, and G. SCOTT. 1972. Morphology, isopycnic density and lipid content of synaptic complexes isolated from developing cerebellums and different brain regions. Bruin Res. 37: 53-68. 13. KORNGUTH, S., A. FLANGAS, J. PERRIN, R. GEISON, and G. SCOTT. 1972. Iso!ation of synaptic complexes in a CsCl gradient: conditions for maximal resolution in
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14.
15.
16.
17. 18.
19. 20. 21. 22. 23. 24. 25. 26.
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ET
AL.
the zonal rotor B-XIV and circular dichroism patterns. Prep. Biochem. 2: 167-192. KORNGUTH, S. E., A. FLANGAS, F. SIEGEL, R. GEISON, J. O’BRIEN, C. LAMAR, and G. SCOTT. 1971. Chemical and metabolic characteristics of synaptic complexes from brain isolated by zonal centrifugation in a cesium chloride gradient. J. Biol. Chem 246 : 1177-1181. KORNGUTH, S., B. WANNAMAKER, E. KOMDNY, R. GEISON, G. SCOTT, and J. O’BRIEN. 1974. Subcellular fractions from Tay-Sachs brains : Ganglioside, lipid, and protein composition and hexosaminidase activities. J. Neurol. Sci. 22: 38346. LENANDOWSKI, L., F. LIEF, M. VERINI, M. PIENKOWSKI, V. TERMEULEN, and H. KOPROWSKI. 1974. Analysis of a viral agent isolated from multiple sclerosis brain tissue: Characterization as a parainfluenza virus, Type I. J. V&Z. 13: 1037-1045. NAFCANG, H. K. and E. J. FIELD. 1973. An electron-microscopic study of multiple sclerosis biopsy material : some unusual inclusions. J. Neural. Sci. 18 : 287-300. NORRBY, E., H. LINK, J.-E. OLSSON, M. PANELIUS, A. SALMI, and B. VANDVIK. 1974. Comparison of antibodies against different viruses in cerebrospinal fluid and serum samples from patients with multiple sclerosis. Infect. Zmmun. 10: 688-694. PRINEAS, J. 1972. Paramyxovirus-like particles associated with acute demyelination in chronic relapsing multiple sclerosis. Science 178: 760-763. RAINE, C., J. POWERS, and K. SUZUKI. 1974. Acute multiple sclerosis: confirmation of paramyxovirus-like intranuclear inclusions. Arck. Neural. 30: 39-45. SUZUKI, K., J. ANDREWS, J. WALTZ, and R. TERRY. 1969. Ultrastructural studies of multiple sclerosis. Lab. Iwest. 20: 444-454. WANNAMAKER, B. B. and S. KORNGUTH. 1973. Electrophoretic patterns of proteins from isolated synapses of human and swine brain. Biochim. Biophys. Acta 303 : 333-337. WANNAMAKER, B. B., S. KORNGUTH, G. SCOTT, A. DUDLEY, and A. KELLY. 1973. Isolation and ultrastructure of human synaptic complexes. J. Neurobiol. 4: 543-55s. WATERS, D. J., and R. H. BUSSELL. 1974. Isolation and comparative study of the nucleocapsids of measles and canine distemper viruses from infected cells. Virology 61: 6479. WEBER, K., and M. OSBORN. 1%9. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 224: 440&4412. WHITTAKER, V. P. 1965. The application of subcellular fractionation techniques to the study of brain function, pp. 39-%. Iti “Progress in Biophysics and Molecular Biology,” Vol. 1.5. J. A. V. Butler and H. E. Huxley, [Eds.]. Pergamon, Oxford.
Isolation
S.
48, 231-240
NEUROLOGY
(1975)
of Synaptic Complexes from Frozen and Thawed Tissue Affected by Multiple Sclerosis U.
KORNGUTH,
J. Departnke?kfs U&persity
of
VARAKIS,
JUHL,
T. D.
G.
JOHNSON, FUCCILLO,
SCOTT,
AND
J.
L. SEVER
Brain
KNOBELOCH, 1
Neurology,
of bzlisconsin National
Physiological Chemistry, Akkatonky, akad Pathology; Medical CePkter 53706, a& the Ixfcctious Discase Branch, Iastitute of Neurological D&use and Stroke, NIH, Bethesda, Maryland 20014 Receizfed
Febrzkary
20, 1975
The report compares the characteristics of isolated synaptic complexes (axon terminals retaining adhesion to the postsynaptic membrane) from frozen and unfrozen human brains (three from multiple sclerosis patients). All brains were obtained postmortem. The buoyant density in CsCl sucro’se density gradients of the synaptic complexes from frozen or fresh brain were identical (p 1.18-1.19) to each other and to the density of synaptic complexes from other humans and other species. Isolated synaptic complexes were readily recognizable by electron microscopy after freezing and thawing of the tissue. The membrane protein composition and buoyant density of the isolated synapses from frozen-thawed brains of patients who died of glomerular nephritis or multiple sclerosis were identical to each other in these parameters and to synapses from unfrozen brains. These fiindings indicate that frozen human brain may be used for several studies involving isolated synapses.
INTRODUCTION Intact synaptic junctions have been successfully isolated from fresh brain of a variety of species (1, 5-7, 11, 13, 14, 26) including man (23). When CsCl-sucrose gradients were used for isolation of synaptic endings, the equilibrium buoyant density of these intercellular organelles was 1.1751.190 whether the tissue used was from guinea pig (ll), swine (12-14)) 1 This work was supported by contract No. NS4-2308 and Grant NS 5631 from NINDS and by a gift from Dr. and Mrs. Leonard Weiss, Madison, Wisconsin. The authors express their appreciation to Dr. G. ZuRhein of the Department of Pathology, University of Wisconsin Medical Center for dissection of the tissue and advice in preparation of the manuscript. 231 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form re.wved
232
KORNGUTH
ET
AL.
monkey (14) or man (15, 23). The buoyant density of the synaptic endings was not affected either by the stage of ontogeny of the animal (12, 15, 23) or by the existence of various neuropathological states in man including hamartoma, Menke’s Kinky Hair syndrome, dementia, and various tumors (15, 23). The only disease observed to cause an alteration in the buoyant density of synapses was Tay-Sachs disease (15). In all of these above studies, unfrozen tissue was used (obtained within 20 hr postmortem). For the investigator interested in studying brains from diseased subjects, it is not always possible to obtain fresh tissue. For example, although fresh brain tissue from patients who died with multiple sclerosis is not readily available, there is an excellent bank of frozen brain specimens (collected by Dr. W. Tourtellotte). The buoyant density of synapses from multiple sclerosis tissue was of particular interest because of the reports that serum from multiple sclerosis patients affects synaptic transmission (3, 4) and the further observation (17) that unusual tubules (18 nm in diameter) were present in postsynaptic regions of six such patients. If these tubules were indeed viruslike structures containing mucleic acid, the buoyant density of the synaptic material might be increased. In this report it will be shown that well preserved synaptic complexes can be readily recovered from frozen brain tissues and that the buoyant density of synapses is 1.18-1.19 which is typical of synapses from other human brains (15, 23). Synapses from frozen multiple sclerosis samples also had normal buoyant densities. MATERIALS
AND
METHODS
To evaluate the effect of freezing and thawing on the isolation and buoyant density of synapses, sections of the frontal and temporal lobes were obtained three hours postmortem from a 13 year old female who died with autoimmune glomerular nephritis (Goodpasture syndrome). A portion of each region was processed immediately for isolation of synapses, and a second portion was frozen at -80 C for 24 hr prior to study. Gross and microscopic examination of this brain revealed no distinct cortical lesions. Frozen samples of brains from an individual who died with multiple sclerosis was obtained from the West Allis Memorial Hospital (with the cooperation of Ms. Lucille Uhlig, Multiple Sclerosis Society of Milwaukee) and two from the multiple sclerosis brain collection of Dr. W. Tourtellotte (University of California at Los Angeles), The specimens had been frozen for at least 1 year prior to study. Tissue of frontal and occipital lobes was used for isolation of synapses. After thawing at room temperature (22 C) for 30-60 min, cerebral white matter was dissected by scalpel away from the gray matter and the pial
SYNAPTIC
COMPLEXES
233
membrane was stripped off. Only the gray matter itself was processed for isolation of synapses. The white matter from all samples was used in other experiments not described in this report. Small portions of each brain were fixed intact to permit comparison of subcellular elements with structures existing in ritzy. The pieces (+ 27 mm”) were placed in cold cacodylate-buffered (0.1 ~1, pH 7.2) glutaraldehyde (2.5%) for 30-45 min. The tissues were rinsed for 15 min in the same buffer without glutaraldehyde, postfixed for 1 hr with 1% 0~04 in the same cacodylate buffer, and processed for embedding in Araldite. Isolation of Synaptic Complexes. The dissected gray regions from the cases with autoimmune glomerular nephritis and multiple sclerosis were placed immediately in sucrose solution A (0.32 M sucrose, 0.001 M MgClz and 0.0004 M phosphate buffer, pH 7.0). The tissue was homogenized in 5 vol of sucrose solution A with a Dounce tissue grinder. The homogenate was centrifuged at 1lOOg for 20 min and the resultant supernatant was decanted and centrifuged at 11,OOOg for 40 min. The ll,OOOg pellet was suspended in sucrose solution A (for each gram of original gray matter used there were 8 ml of suspending sucrose solution A). An aliquot of this suspension (8.32 ml) was mixed with 1.68 ml saturated CsCl (previously adjusted to pH 7.0) and then placed in polyallomer tubes for use in the SW 41 Beckman rotor. Density gradient beads (Beckman Co.) for the range of 1.10 and 1.20 g/cm3 were added to each tube. The density gradient was generated during a 66 hr centrifugation at 36,000 rpm in the LS-50 ultracentrifuge with the SW 41 rotor. After centrifugation, each visible band was removed by puncturing the polyallomer tube with a needle (20 gauge) and withdrawing the band into a syringe. The bands were diluted with 2 vol of sucrose A and centrifuged at 100,OOOg for 1 hr in the 50 rotor. Portions of the pellets were processed for electron microscopic analysis and the remainder used for biochemical analysis. Electvorz Il~icvoscopy. Pellets were fixed for 1 hr in 1% 0~04 buffered with 0.1 M cacodylate (pH 7.2) and processed for embedding in Araldite. Thin sections were contrasted with uranyl acetate and lead citrate. They were examined and photographed either in an AEI 801 or Philips EM-200 electron microscope. Electrophoresis of Proteins. The fractions recovered from the CsCl-sucrose gradient were lysed by suspension in 10 vol of distilled water. The lysate was centrifuged at 10,OOOg for 40 min and this procedure repeated two additional times. The final insoluble membrane fractions were solubilized with 1% sodium dodecyl sulfate. The resultant solutions were subjected to electrophoresis by the method of Weber and Osborn (25) for 18 hr on 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate and 0.1 M phosphate buffer (pH 7.0) using a constant current of
234
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ET
AL.
3 ma per gel. Gels were then stained with Coomassie blue for 1 hr and destained in 5% methanol and 9.2% acetic acid in water. Gels were photographed and stored in an aqueous solution of 7.5% acetic acid. RESULTS Isolation of tlze Synaptic Complexes. Each of the fractions from unfrozen gray matter and frozen gray matter were similar with one exception. The 1lOOg pellet obtained from the homogenate of fresh ,tissue had three layers : (i) a discrete pink zone at the tube bottom which contained primarily nuclei ; (ii) a loose white layer above the pink ; (iii) a white filmlike layer on top. The pink nuclear zone had sufficient cohesiveness to permit its isolation from the above two layers. The 1lOOg pellet recovered from the frozen tissue had no discrete nuclear zone although the lower portion of the pellet had a pink hue. Although intact nuclei could be readily identified by phase microscopy of the pellet from frozen brain, they were mixed in with other cell components to a much greater extent than in the pink zone of unfrozen brain pellets. After centrifugation of the crude mitochondrial fraction (11,OOOg pellet) in the CsCl-sucrose gradient, the banding pattern of material from fresh and frozen brain samples appeared very similar (Fig. la, b). There is a band (A) at density 1.10 which contains myelin as determined by ultrastructural study. A second band (p 1.14-1.16) (B) has the majority of particulate material and a third band (C) 1.18-1.19 is apparent above the 1.2 density marker bead. A fourth granular band (D) appears below the 1.2 marker. The banding pattern in the CsCl sucrose gradient of the material from the multiple sclerosis brains (Fig. lc) resembled that of the patient with autoimmune glomerular nephritis with two exceptions. Zone B from the multiple sclerosis tissue was much reduced in amount and was distributed over a larger density region (from 1.13 to 1.17) compared to a distinct band B from the autoimmune glomerular nephritis brain. Band D from the multiple sclerosis material was at 1.20-1.21 and was more compact than band D from the autoimmune glomerular nephritis disease. Band C in both cases ranged between 1.18-l. 19 (this band contained intact synaptic endings and is described below), The tube c was drawn from the fractionation of one of the multiple sclerosis patients. Patterns from the other two multiple sclerosis patients were identical to this illustrated pattern. Electron Microscopy. Synapses were readily identified in the frozenthawed tissue prior to homogenization (Fig. 2 insets) and after homogenization and centrifugation (Fig. Za, b). The majority of all synaptic complexes recovered from the CsCl sucrose gradient were localized in band C, and the major components present in band C were synaptic end-
S17NAPTIC
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b
FIG. 1. Distribution of membranes from unfrozen control, frozen control and frozen multiple sclerosis tissue in the cesium chloride density gradient. The fraction applied to the gradient was from the 11,OOOg pellet resuspended in sucrose A. Prior to centrifugation 8.32 ml of the suspension was mixed with 1.68 ml of saturated CsCl solution and the mixture was centrifuged for 66 hr. The filled circle at the region of band A represents the density gradient bead (p 1.10) and the filled circle by band C or D represents the density gradient bead (p 1.20). Tube a is from the unfrozen control brain, tube b is from the frozen and thawed control brain; tube c is from the multiple sclerosis case. Band C contained the isolated synaptic complexes ; electron microscopic photographs of these synaptic complexes are shown below the drawing of each tube. The presynaptic region is desi,gnated s. (X 28,050).
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FIG. 2. Electron micrographs of membranes in band C recovered after fractionation of the brains obtained from patients who died with multiple sclerosis. Figure Za is from one patient and Fig. 2b is a sample from a second patient. Both tissues were obtained in a frozen condition prior to study. Several isolated synaptic endings are designated S. Magnification is 30,400 for a and 27,600 for b. The insets are photographs
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ings (Fig. 2). After freezing and thawing of human brain tissue prior to homogenization the presynaptic and postsynaptic elements were readily identifiable (Fig. la, b, c) and retained their adhesion. Of particular interest was the finding that intact synapses could be isolated from multiple sclerosis brains (Fig. 2) that had been frozen at -80 C for extended periods (longer than 1 year) and that these synaptic complexes had a buoyant density typical of synapses prepared from unfrozen human brains ( 1.175-l. 19). Membrane-enclosed glycogen granules were observed in band C as previously reported (11) for guinea pig brains fractionated in CsCl density gradients. Narang and Field (17) reported tubular arrays in postsynaptic regions of multiple sclerosis brains and suggested that these arrays may be viruses or neurotubules. In the present study, clear tubular arrays were not seen in any fraction, This may reflect either absence of 18 nm tubular structures in the brains examined or destruction of their ultrastructure by the freeze-thaw procedure similar to that seen after freezing of measles virus (24). Band A from the autoimmune glomerular nephritis patient contained primarily myelin fragments with the usual appearance. Band A from the multiple sclerosis brains however contained lysosomes and degenerative myelin in addition to myelin. Band B contained many membraneous fragments of undefined origin and fragments of smooth endoplasmic reticulum. Band D contained free mitochondria, filamentous material and chromatinlike fibrils. Chromatin was most likely released from the nuclei ruptured by the freezing process (evidenced by loss of the discrete pink zone described above). Electrophoretic Analysis of Synaptic Proteins. The protein components of the synaptic membranes recovered from the CsCl-sucrose gradients are shown in Fig. 3. Synapses from multiple sclerosis brains and autoimmune glomerular nephritis brains had similar protein patterns on polyacrylamide sodium dodecyl sulfate gels. The major protein species have molecular weights 92,000, 53,000, and 43,000. The protein analysis on polyacrylamide gels reveals a similar molecular weight distribution of proteins in synapses from patients having either multiple sclerosis or autoimmune glomerular nephritis. This protein pattern is similar to that of synapses from other human brains ( 15,22). DISCUSSION This report shows that the process of freezing does not modify the buoyant density of isolated synaptic complexes and that these intercellular of synapses present in the frozen-thawed brains fixed prior to homogenization and centrifugation. Each inset is of the brain sample used for isolation shown in Fig. 2a and b. Inset a is 28,178 X and b is 28,730 X.
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patterns of proteins in isolated synaptic complexes from the FIG. 3. Electrophoretic control brain, the multiple sclerosis brain and swine brain. The membranes in fraction C from the CsCl gradients were solubilized with 1% sodium dodecyl sulfate and subjected to electrophoresis for 18 hr on 10% polyacrylamide gels (0.1 M phosphate buffer, pH 7.0) containing 0.1% sodium dodecyl sulfate. The current was 3 ma per gel with the cathode at the top and anode at the bottom. Gels were stained with Coomassie blue for 1 hr and destained in methanol: acetic acid. Gel a is of the swine proteins, b of unfrozen controls, c of frozen controls and d of multiple sclerosis fractions. The protein pattern from each gel is similar to that of the other gels.
organelles can be recognized in the electron microscope. Additionally, synapses from brain tissue affected by a prolonged condition of multiple sclerosis have buoyant densities (1.18-1.19) in CsCl and an ultrastructure and protein pattern that is identical to those of synapses from other species (11, 14) and other human brains except from Tay-Sachs disease (15,22, 23). The observation that frozen-thawed brain can be used for certain ultrastructural and biochemical investigations of synaptic elements should facilitate research efforts with respect to neural disorders. The researcher can use brain samples kept in a frozen state for several years or can receive frozen
samples
from
distant
laboratories.
The findings that synapsesfrom multiple sclerosis brains have the same buoyant density as synapsesfrom other brains is interesting becauseof the postulated role of slow viruses in multiple sclerosis (2, S-10, 16-21). Only
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one of these reports (17) suggests that viruses may be localized near synapses whereas the others involve the region adjacent to the plaque. The subcellular components containing aggregates of viruses may be expected to have an increased buoyant density because of the high density of nucleic acid (161.7). None of the fractions from gray matter (including synapses) of the multiple sclerosis tissues examined have an increased buoyant density, and on this basis it appears that synaptic complexes isolated from multiple sclerosis brain do not contain high concentration of nucleocapsid. In summary, we conclude that multiple sclerosis does not cause the appearance of membrane-associated synaptic proteins having unusual molecular weight distributions and that the freezing process does not markedly affect the buoyant density or the protein composition of synaptic components. REFERENCES 1. ABDEL-LATIF, A. A. 1961. A simple from rat brain. Bioclzinr. Biophys. 2. ADAMS, J. M., and D. T. IE*IAGAWA.
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