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Ultrastructural aspects of discs in rod outer segments
Exp. Eye Res. (1973) 16, 173-182
Ultrastructural
Aspects of Discs in Rod Outer Segments IZHAK NIR AND DANIEL C. PEASE
Lkpartment
of Anatomy,
University
of Calijornia,
Los Angeles, Cal$. 90024, U.S.A.
(Received 13 November 1972, and in revised form 5 March 1973, Boston)
Cytomembrane lipids are retained in tissue embedded in polymerized glutaraldehyde-urea, and a collapse of hydrated carbohydrate gels is prevented. After this embedment the discs of retinal rod outer segments demonstrated persistent 15-20 A-wide intradisc spaces even when exposed to extremely hypertonic environments. On the other hand, conventional dehvdration and embedding in Araldite eliminated these spaces, resulting in the pentalamkar patterns of arrangement previously recognized. The latter are regarded as artifactually created as a result of the collapse of the supporting gel in the final stages of conventional dehydration. Persistent intradisc spaces are in agreement with X-ray diffraction measurements which suggest about 25 B intradisc gaps in fresh outer rod segments. It, was serendipitously found that the disc membranes and inner rod segment mitochondria remained osmotically active after an aldehyde fixation, so that subsequently the size of the intradisc spaces and mitochondria varied with osmotic manipulation. In order to preserve these spaces as they existed under particular osmotic conditions, a secondary osmium tetroxide fixation in an osmotically controlled medium proved to be necessary.
1. Introduction Retinal rod outer segments appear to consist of isolated (Cohen, 1968, 1970), flattened discs, stacked in rows at right angles to the incident light. An accurate understanding of the organization of the discs seems imperative if the relation between structure and function is to be properly evaluated. In this regard a remaining question is whether or not the two membranes which form a disc are in immediate contact, or whether a true intradisc space exists under normal physiological conditions. The evidence which favors direct membrane contact is based primarily on conventional electron microscopic preparations which demonstrate pentalaminar images when the discs are observed in transverse sections (Moody and Robertson, 1960; FerngndezMorBn, 1961; Nilsson, 1965; and Dunn and Adornian? 1971). On the other hand, results of X-ray diffraction studies have suggested that a hydrated compartment exists between the disc membranes in native outer segments (Blaurock and Wilkins, 1969; and Worthington, 1971). :Recently, Peterson and Pease (1970) and Pease and Peterson (1972) introduced a new, water-containing embedment which obviates the need for full dehydration. This is based upon polymerizing 50% glutaraldehyde with urea under acidic conditions. Advantages of the method include the preservation of membrane lipids in sectioned material, and also the retention of those spatial relationships which are maintained in life by carbohydrate gels. Thus, it seemed appropriate to explore the value of this technique in an investigation aimed at resolving questions of disc ultrastructure. We believe that we have succeeded in demonstrating that the suspected functional intradisc space can be preserved and visualized in spite of osmotic manipulation, with either NaCl or sucrose, and so it represents a distinct compartment. *
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Basically our experiments on frog rod discs fell into three groups, designed to compare the effects of decidedly hyper- and hypotonic exposures with an isotonic enviromnent. Additionally, retinas embedded in 50% glutaraldehyde polymerized with urea were cornpared with conventionally dehydrated and embedded material. Frogs kept in the dark for 12 hr preceding the experiments were used. In one series of experiments, incubation (osmotic manipulation), fixation and embedding were carried out under normal conditions of illumination. In another series, these steps were completed in dim red light. No differences were apparent. Experiments began by decapitating the frogs. The eyes were enucleated and the anterior part was removed and discarded. The posterior part with the sensory retina was then flushed in a large quantity of an incubation medium, and quickly transferred to fresh incubation medium for an 8-min interval before fixation. Then, in order to minimize the possibility of mechanical damage, an aldehyde fixation preceded further dissection. We regard it as very important to emphasize that in any given experiment, the tonicity of the incubation medium, and all fixative and wash solutions, were matched as far as was reasonably possible and deemed necessary. An osmometer was used to monitor all solutions. As indicated below, a Na-phosphate buffer was used with all solutions. Heller, Oswald and Bok, (1971) have demonstrated that disc membranes are impermeable to this substance, and that it exerts an osmotic effect comparable with NaCl. Severely hyperosmotic solutions were prepared in two ways, either by adding NaCl or sucrose to a phosphate buffer. First, with NaCl, for incubation and rinses, 0.1 M Naphosphate buffer+l% NaCl was used; for glutaraldehyde fixation, 1% glutaraldehydef 0.05 M Na-phosphate bufferfly NaCl; f or osmium tetroxide postfixation, l”//o OsO,+0.1 M Na-phosphate buffer+l% NaCl. S econd, with sucrose, for incubation and rinsing, 0.1 M Na-phosphate buffer+lO”/o sucrose; for glutaraldehyde fixation, 1% glutaraldehyde +0*05 M Na-phosphate buffer+lO% sucrose; for osmium tetroxide postfixation, 1s’” OsO,+O.l M Na-phosphate buffer+lO% sucrose. The total tonicity of these solutions, determined by freezing-point depression measurements: was between 530-580 mOsmo1. Cohen (1971) has indicated that a pressure of 225 mOsmo1 corresponds to the normal environment of the frog retina. Therefore, approximately isotonic solutions were prepared as follows, with total osmotic pressures of 200-240 mOsmo1. For incubation and rinses, 0.1 M Na-phosphate buffer was used; for glutaraldehyde fixation, 1% glutaraldehyde-+ 0.05 M Na-phosphate buffer; for osmium tetroxide postfixation, l”/b OsO,+O.l M Naphosphate buffer. It is appreciated that glutaraldehyde may enter cells so freely and rapidly that it does not exert a functionally important tonic effect. Thus, in its presence, it is hard to decide what should be regarded as an isotonic solution. This is largely a moot’ question, however, when qualitative effects of decidedly hypo- and hypertonic solutions are to be compared, as in what follows. This is particularly so, as will be seen, when it is appreciated that the preserved morphological patterns reflect osmotic circumstances at the time of secondary 0~0, fixation, rather than during primary glutaraldehyde treatment. Decidedly hypotonic solutions with osmotic values ranging between 50 and 115 mOsmo1 were prepared as follows. For incubation and rinses, 0.02 M Na-phosphate buffer was used; for glutaraldehyde fixation, 1% glutaraldehyde+0.005 M Na-phosphate buffer; fol osmium tetroxide post-fixation, 1% OsO,+O.O2 M Na-phosphate buffer. Buffering was always at pH 7.0. Incubation was for approximately 8 min, accompanied by continuous shaking. This was followed by glutaraldehyde fixation, carried out at room temperature for 150 min. After about 120 min the retina was teased away from the underlying choroid and cut into small pieces. A lo-min rinse with the incubation medium followed the glutaraldehyde fixation. Then, generally, the pieces of tissue were transferred to the osmium tetroxide fixative,
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also with an appropriately adjusted tonicity. This fixation was continued at room temperature for 60 min, and was followed by an additional wash in the original incubation medium. The tissue samples were then divided at random into two groups, one for embedding in the glutaraldehyde-urea resin as described below, and the other for Araldite, following a conventional alcoholic dehydration, and the use of propylene oxide as a transitional solvent. Some tissue samples were embedded without the osmium tetroxide postfixation but then, as will be seen, the membranes retained osmotic capabilities for at least a part of the infiltration process. -4s a prelude to embedding in glutaraldehyde-urea, the concentration of the glutaraldehyde was increased stepwise. First, the tissue was transferred into 5% glutaraldehyde in 0.05 M Na-phosphate buffer, pH 7.0. After 15 min the glutaraldehyde concentration was increased slowly by adding successive quantities of 50% glutaraldehyde with its pH adjusted to 4.1. In one set of experiments with hypertonic manipulation, 1% NaCl was added to the glutaraldehyde. The exchange into 50% glutaraldehyde was completed within 30 min. At this stage the tissue was transferred into a glutaraldehyde-urea mixture preparatory to polymerization. The glutaraldehyde-urea embedding procedure has been described elsewhere (Pease and Peterson, 1972). Basically, it consists of preparing in advance a very strong solution of urea dissolved in a minimum quantity of 50% glutaraldehyde, with a carefully adjusted acidic pH. This mixture is stable for a few days at room temperature. Just before use it is diluted with an appropriate additional amount of 50% glutaraldehyde, also with an adjusted pH. At room temperature the mixture begins polymerizing in less than 30 min at a pH of about 4.3. In so far as longer infiltration times seem desirable, tissue is moved through successive changes of freshly blended mixtures. For the present series of experiments, two parts of the stable glutaraldehyde-urea solution were blended with one additional part of glutaraldehyde. Infiltration involved two changes of freshly prepared 2 : 1 mixtures at 15-min intervals, after which the tissue blocks were isolated in small drops of the resin as the mixture began to thicken. The blocks were left to cure at room temperature over a two-night period. Finally, the blocks were mounted with white carpenter’s glue on supports consisting of short lengths of appropriately thick dowel-rod, with tips shaped in a pencil sharpener as truncated cones. Glass knives were used for sectioning, at substantially higher speeds than are conventional. Generally, and unless otherwise noted, sections were stained first with uranyl acetate, and then with lead citrate. However, in order to visualize membranes with positive contrast when postfixation with osrnium tetroxide had not been employed preceding a glutaraldehyde-urea embedment, such sections were exposed to 0~0, vapors. This then COJIstit,uted their only “stain”. 3. Results Pilot experiments indicated that a secondary fixation with osmium tetroxide was required if disc dilatations induced by hypotonic solutions were to be preserved throughout a glutaraldehyde-urea embedment. Otherwise, if only a glutaraldehyde fixation was used, the membranes retained osmotic properties, and swollen discs returned to normal configurations as the glutaraldehyde (or glutaraldehyde-urea) content was rai.sed preparatory to the embedment. A comparison of the first two figures will make this evident, for (aside from staining) both preparations experienced identical treatment excepting only that the tissue of Fig. 2 was postfixed with OsO,, whereas that of Fig. 1 was not. Figure 2 demonstrates the substantial disc swelling and extreme mitochondrial enlargement that might logically be anticipated as a consequence of . . hypotonm mcubation. The swelling was substantially consistent in a given outer segment, and from one preparation to another. However, the swellings are not seen
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at all in the tissue illustrated in Fig. 1, even though these organelles ntlist ~urel! have been similarly enlarged at one time, and to have been swollen even during t8hcA interval of their glutaraldehyde fixation. Evidently all traces of the enlargement,< disappeared, presumably as thematerial, was infiltrated subsequently, with increasing concentrations of glutaraldehyde (or glutaraldehyde-urea mixture) which inevitabl! eventually led to hypertonic conditions being established. Heller: et al. (1971) also have indicated that disc swelling can be induced or reversed after glutaraldehydc fixation by changing the osmolarity of postfixation media. Retinae which were treated with solutiolis in the iso-osmotic range, were postfixed
FIG. 1. Retina incubated in a decidedly hypotonic solution, and then fixed with hypotonic glutsraldehyde. Subsequently, the retina wa8 exposed to hypertonic conditions a8 a prelude to embedding in polymerized glutaraldehyde-urea. The section ultimately was stained by exposure to 0~0, vapor. Sotr normal looking mitochondria a8 well as highly ordered discs. ( x 34,340)
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with 0~0, and were then embedded in polymerized glutaraldehyde-urea, demonstrated fairly well-organized arrays of discs (Fig. 3). Intradisc spaces always were evident. The width of these spaces was somewhat variable, presumably reflecting small osmotic inequalities. The minimum width approached 20 A and was an ordered feature of many areas. Wider intradisc spaces lacked order and specific dimensions, but generally did not exceed 30 .& in width. Individual nienibranes clearly showed a trilitminar pattern. Retinae exposed to decidedly hypertonic solutions which subsequently were embedded in polymerized glutaraldehyde-urea invariably demonstrated very welldefined and uniformly wide intradisc spaces. It made no essential difference whet,her
Pm. 3. Retina treated exactly 080, solution before embedding Note greatly dilated mitochondria,
as in Fig. 1, except in glutaraldehyde-urea. as well a8 swollen
that it was secondarily fixed with a hypotonic Stained with uranyl acetate and lead citrate. and disordered discs. ( x 39,000
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the original exposure was to hypertonic NaCl (Fig. 4) or sucrose (Fig. 6). The. witkh of these spaces approximated 15-20 A, although the gap seemed slightly (- 5 A) wider after NaCl incubation (Fig. 4) than after sucrose (Fig. 6). We believe t,hat this demonstration of an intradisc compartment even under conditions of extreme hypertonicity is a reflection of our improved embedding technique. These same preparations also demonkrated a kilaminar organization of individual
FIG. 3. Retina first incubated, and then sequentially fixed with glutaraldehyde and in isotonic solutions. The retina was embedded in polymerized glutaraldehyde-urea, stained with uranyl acetate and lead citrate. ( x 160,000) FIG. 4. Retina incubated, and then fixed sequentially with glutaraldehyde and 080, hypertonic with N&I. The retina was embedded in polymerized glutarsldehyde-urea stained with uranyl acetate and lead citrate. ( ,-. 160,000) FIQ. 5. Retina treated exactly as for Fig. 4, except t,hat it w&s conventionally embedded in Araldite. The section was stained with uranyl acetate and lead citrate.
osmium and
tetroxide, the section
in solutions and the dehydrated ( ‘i 160,000)
made section and
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membranes. This was particularly evident when the hypertonicity had been established by using NaCl (Fig. 4). When sucrose was used to create hypertonic solutions, the trila,minar patterns then were less evident (Fig. 6).
FIG. 6. The retina was first incubated and then sequentially fixed with glutsraldehyde and 0~0, in solutions made hypertonic with sucrose. It was then embedded in polymerized glutaraldehyde-urea. The section was stained with uranyl acetate and lead citrate. ( x 240,000) I’m. 7. Retina treated exactly as for Fig. 6, except t.hat it was conventionally dehydrated and embedded in Araldite. The section was stained with many1 acetate and lead citrate. ( ~240,000)
The apparent width of the disc membranes demonstrated some dependence on the means used to create hypertonicity. After exposing retinae to hyperosmotic NaCl, and embedding in polymerized glutaraldehyde-urea, the total disc width was about 210 8. When sucrose was used instead of NaCl, the corresponding width was only
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about 165 A. Thus, in subtle ways, the pattern of preservation of membrane structure was influenced by the character of the vehicle used with the fixative. When conventional techniques of dehydration and embedding in Araldite were employed, significant detail was lost. This is evident in Fig. 5 where the intradisc space was not discernible as a separate entity after incubation with hypertonic NaCI. Instead, there was then the appearance of fused membranes so that the full width of each disc appeared to have a pentalaminar pattern of organization such as described previously by Moody and Robertson (1960) Fernandez-Moran (1961), Nilsson (1965), and by Dunn and Adomian (1971). Th us, under these circumstances, it was not generally possible to differentiate what may be called the “inner leaflets” of the disc
FIG. 8. Retina first incubated and then fixed only with glutaraldehyde with NaCl. The glutaraldehyde-urea embedding mixture also contained stained by exposure to 0~0, vapor. (X 118,000)
in a solution made hypertonic 1 “/f NaCI. The section was finally
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membranes from the disc spaces. Furthermore, the “outer leaflets” were not well preserved and differentiated either. After hypertonic sucrose incubation even less structural pattern was retained through conventional dehydration and embedding in Araldite (Fig. 7). A question remains as to whether or not postfixation with osmium tetroxide is rea.lly essential for the demonstration of the intradisc spaces under hyperosmotic conditions. To investigate the possibility that the appearance might be artifactually dependent on osmium tetroxide, retinae exposed to hypertonic media were fixed only in .hypertonic glutaraldehyde, and then embedded in polymerized glutaraldehydeurea without 0~0, postflxation. In some experiments, 1% NaCl was even added to the embedding mixture to insure the maintenance of a high salt concentration. Thin sections of such material are known to retain their membrane lipids which then can be stained with osmium tetroxide vapors (Pease and Peterson, 1972; Peterson and Pease, 1972). The results of these preparative procedures are shown in Fig. 8. The intradisc space is clearly evident, and thus its persistence and demonstration are independent of an osmium tetroxide fixation. Furthermore, it should be noted that there was no essential difference evident in disc morphology between this material, prepared under hyperosmotic conditions, and that illustrated in Fig. 1 which originally was exposed to a hypotonic medium. It is also seen in Figs 1 and 8 that when 0~0, was not used as a fixative, but rather as a stain, the disc membranes looked thinner tha,n otherwise, and were without evidence of trilaminar patterns. Correspondingly, the disc spaces seemed wider than after postfixation and conventional staining. Thus, it would seem that 0~0, vapors stain somewhat different membrane components tha,n do uranyl acetate and lead citrate. This agrees with findings relative to myelin (Peterson and Pease, 1972) and chloroplast membranes (Nir and Pease, 1973), and confirms what Godfrey (1972, 1973) has seen and measured in rod disc membranes. 4. Discussion The most important part of our experiments involved the osmotic manipulation of rod outer segment discs to see whether under any circumstances the intradisc spaces could be obliterated if the preservation technique was adequate. Since these spa,ces were found always to persist after a glutaraldehyde-urea embedment, it was also deemed necessary to compare results with standard preparative techniques to understand the previous failures to observe these spaces that have been recorded in the literature (Moody and Robertson, 1960; Fernandez-Moran, 1961; Nilsson, 1965; and Dunn and Adomian, 1971). We conclude that conventional procedures do not adequately preserve intradisc spaces and their contents. Godfrey (1972, 1973) has already indicated that the contents of the intradisc spaces are stained with acidic silicotungstic acid which has led him to propose that carbohydrate moieties are to be found there. The spaces and their staining characteristics are comparable to relationships we have established in studying the intraperiod complex of myelin (Peterson and Pease, 1972) and “glycocalyx” in other situations (Pease and Peterson, 1972). Thus, there is comparative evidence that the intradisc spaces may represent volumes occupied in life by hydrated carbohydrate gels. Of course, considering that rod discs originate by invagination of the plasma membrane, it is reasonable to consider homologizing their contents with surface coats. A great advantage of the glutaraldehyde-urea embedment is that polymerization occurs, and spatial relationships become fixed, while there is still substantial water
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in the medium. Thus, highly hydrated gels need not be collapsed as probably often occurs in the final stages of conventional dehydration, as the last of the water is removed (Pease and Peterson, 1972). Gel collapse could most plausibly explain the obliteration of the disc cavities and the appearance of pentalaminar patterns in outer segments prepared by usual procedures (Moody and Robertson, 1960; Fern&ndezMoran, 1961; Nilsson, 1965; and Dunn and Adomian, 1971). Measurementsthat have beenderived from X-ray diffraction studiesof fresh retinae have suggestedthat 5-25 A-wide intradisc compartments exist in life (Blaurock and Wilkins, 1969; Worthington, 1971). The larger figure agreesreasonably well with our own finding that the minimal spacesthat survive exposure to undoubted hypertonic solutions are about 15-20 A wide. When we attempted to approximate isotonic conditions, in many places the compartments were no wider than this. Thus, the evidence suggeststhat under normal and stable physiological conditions, this minimal value may pertain. Little or no osmotic swelling need be anticipated in life. Disc swelling in a hypotonic medium is, of course, a well-known phenomenon (Heller, et al. 1971) even though Cohen (1971) has argued that interpretation may be complex. Our own experiments demonstrate a close parallelism in what is seenin outer segment discs and adjacent inner segment mitochondria. There is no question but that the latter are osmometers. Thus, the results emphasize a morphological pitfall: that an aldehyde fixative must not be relied upon to preserve osmotically-dependent spatial relationships. In essence,at least someof the semipermeable properties and characteristics of disc and mitochondrial membranessurvive moderate concentrations of glutaraldehyde. Membrane-bound volumes can soil1be osmotically manipulated. On the other hand, full permeability apparently is achieved by 0~0, (post-) fixation, which thus provides a convenient way of stabilizing spatial relationships that are osmotically dependent. Although less explicitly stated, this was also one of the conclusions of Heller, et al. (1971). ACKNOWLEDGMENTS
This investigation was supported by U.S. Public Health Service Grant NS 284. REFERENCES Blaurock, A. E. and Wilkins, M. H. F. (1969). Nature (London) 223, 906. Cohen, A. I. (1968). J. Cell Bid. 37,424. Cohen, A. I. (1970). Vision Res. 10,445. Cohen, A. I. (1971). J. Cell Biol. 48, 547. Dunn, R. F. and Adomian, G. E. (1971). I n 29th Proc. Electron Microscopy Sot. Amer., p. 268. (Ed. Arceneaux, C. J.) Claitor’s Pub. Co., Baton Rouge, La. Fernandez-Moran, H. (1961). In The Structure of the Eye, p. 521. (Ed. Smelzer, G. K.) Academic Press, New York and London. Godfrey, A. J. (1972). In 30th Ann. Proc. Electron Microscopy Sot. Amer., p. 50. (Ed. Areeneaux, C. J.) Claitor’s Pub. Co., Baton Rouge, La. Godfrey, A. J. (1973). J. Ultra&u& Res. (In press). Heller, J., Ostwald, T. J. and Bok, D. (1971). J. Cell Biol. 48, 633. Moody, M. F. and Robertson, J. D. (1960). J. Biophys. B&hem. Cytol. 7, 87. Nilsson, S. E. G. (1965). J. Ultrastruct. Res. 12, 207. Nir, I. and Pease, D. C. (1973). J. Ultrastruct. Res. (submitted). Pease, D. C. and Peterson, R. G. (1972). J. Ultrastruct. Res. 41, 133. Peterson, R. G. and Pease, D. C. (1970). In 28th Ann. Proc. Electron Microscopy Sot. Amer., p. 334. (Ed. Arceneaux, C. J.) Cla,itor’s Pub. Co., Baton Rouge, La. Peterson, R. G. and Pease, D. C. (1972). J. Ultrastruct. Res. 41, 115. Worthington, C. R. (1971). Fed. Proc. 30,57.
Ultrastructural
Aspects of Discs in Rod Outer Segments IZHAK NIR AND DANIEL C. PEASE
Lkpartment
of Anatomy,
University
of Calijornia,
Los Angeles, Cal$. 90024, U.S.A.
(Received 13 November 1972, and in revised form 5 March 1973, Boston)
Cytomembrane lipids are retained in tissue embedded in polymerized glutaraldehyde-urea, and a collapse of hydrated carbohydrate gels is prevented. After this embedment the discs of retinal rod outer segments demonstrated persistent 15-20 A-wide intradisc spaces even when exposed to extremely hypertonic environments. On the other hand, conventional dehvdration and embedding in Araldite eliminated these spaces, resulting in the pentalamkar patterns of arrangement previously recognized. The latter are regarded as artifactually created as a result of the collapse of the supporting gel in the final stages of conventional dehydration. Persistent intradisc spaces are in agreement with X-ray diffraction measurements which suggest about 25 B intradisc gaps in fresh outer rod segments. It, was serendipitously found that the disc membranes and inner rod segment mitochondria remained osmotically active after an aldehyde fixation, so that subsequently the size of the intradisc spaces and mitochondria varied with osmotic manipulation. In order to preserve these spaces as they existed under particular osmotic conditions, a secondary osmium tetroxide fixation in an osmotically controlled medium proved to be necessary.
1. Introduction Retinal rod outer segments appear to consist of isolated (Cohen, 1968, 1970), flattened discs, stacked in rows at right angles to the incident light. An accurate understanding of the organization of the discs seems imperative if the relation between structure and function is to be properly evaluated. In this regard a remaining question is whether or not the two membranes which form a disc are in immediate contact, or whether a true intradisc space exists under normal physiological conditions. The evidence which favors direct membrane contact is based primarily on conventional electron microscopic preparations which demonstrate pentalaminar images when the discs are observed in transverse sections (Moody and Robertson, 1960; FerngndezMorBn, 1961; Nilsson, 1965; and Dunn and Adornian? 1971). On the other hand, results of X-ray diffraction studies have suggested that a hydrated compartment exists between the disc membranes in native outer segments (Blaurock and Wilkins, 1969; and Worthington, 1971). :Recently, Peterson and Pease (1970) and Pease and Peterson (1972) introduced a new, water-containing embedment which obviates the need for full dehydration. This is based upon polymerizing 50% glutaraldehyde with urea under acidic conditions. Advantages of the method include the preservation of membrane lipids in sectioned material, and also the retention of those spatial relationships which are maintained in life by carbohydrate gels. Thus, it seemed appropriate to explore the value of this technique in an investigation aimed at resolving questions of disc ultrastructure. We believe that we have succeeded in demonstrating that the suspected functional intradisc space can be preserved and visualized in spite of osmotic manipulation, with either NaCl or sucrose, and so it represents a distinct compartment. *
173
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I.
NIR
AND
2. Materials
D.
C. PEASE
and Methods
Basically our experiments on frog rod discs fell into three groups, designed to compare the effects of decidedly hyper- and hypotonic exposures with an isotonic enviromnent. Additionally, retinas embedded in 50% glutaraldehyde polymerized with urea were cornpared with conventionally dehydrated and embedded material. Frogs kept in the dark for 12 hr preceding the experiments were used. In one series of experiments, incubation (osmotic manipulation), fixation and embedding were carried out under normal conditions of illumination. In another series, these steps were completed in dim red light. No differences were apparent. Experiments began by decapitating the frogs. The eyes were enucleated and the anterior part was removed and discarded. The posterior part with the sensory retina was then flushed in a large quantity of an incubation medium, and quickly transferred to fresh incubation medium for an 8-min interval before fixation. Then, in order to minimize the possibility of mechanical damage, an aldehyde fixation preceded further dissection. We regard it as very important to emphasize that in any given experiment, the tonicity of the incubation medium, and all fixative and wash solutions, were matched as far as was reasonably possible and deemed necessary. An osmometer was used to monitor all solutions. As indicated below, a Na-phosphate buffer was used with all solutions. Heller, Oswald and Bok, (1971) have demonstrated that disc membranes are impermeable to this substance, and that it exerts an osmotic effect comparable with NaCl. Severely hyperosmotic solutions were prepared in two ways, either by adding NaCl or sucrose to a phosphate buffer. First, with NaCl, for incubation and rinses, 0.1 M Naphosphate buffer+l% NaCl was used; for glutaraldehyde fixation, 1% glutaraldehydef 0.05 M Na-phosphate bufferfly NaCl; f or osmium tetroxide postfixation, l”//o OsO,+0.1 M Na-phosphate buffer+l% NaCl. S econd, with sucrose, for incubation and rinsing, 0.1 M Na-phosphate buffer+lO”/o sucrose; for glutaraldehyde fixation, 1% glutaraldehyde +0*05 M Na-phosphate buffer+lO% sucrose; for osmium tetroxide postfixation, 1s’” OsO,+O.l M Na-phosphate buffer+lO% sucrose. The total tonicity of these solutions, determined by freezing-point depression measurements: was between 530-580 mOsmo1. Cohen (1971) has indicated that a pressure of 225 mOsmo1 corresponds to the normal environment of the frog retina. Therefore, approximately isotonic solutions were prepared as follows, with total osmotic pressures of 200-240 mOsmo1. For incubation and rinses, 0.1 M Na-phosphate buffer was used; for glutaraldehyde fixation, 1% glutaraldehyde-+ 0.05 M Na-phosphate buffer; for osmium tetroxide postfixation, l”/b OsO,+O.l M Naphosphate buffer. It is appreciated that glutaraldehyde may enter cells so freely and rapidly that it does not exert a functionally important tonic effect. Thus, in its presence, it is hard to decide what should be regarded as an isotonic solution. This is largely a moot’ question, however, when qualitative effects of decidedly hypo- and hypertonic solutions are to be compared, as in what follows. This is particularly so, as will be seen, when it is appreciated that the preserved morphological patterns reflect osmotic circumstances at the time of secondary 0~0, fixation, rather than during primary glutaraldehyde treatment. Decidedly hypotonic solutions with osmotic values ranging between 50 and 115 mOsmo1 were prepared as follows. For incubation and rinses, 0.02 M Na-phosphate buffer was used; for glutaraldehyde fixation, 1% glutaraldehyde+0.005 M Na-phosphate buffer; fol osmium tetroxide post-fixation, 1% OsO,+O.O2 M Na-phosphate buffer. Buffering was always at pH 7.0. Incubation was for approximately 8 min, accompanied by continuous shaking. This was followed by glutaraldehyde fixation, carried out at room temperature for 150 min. After about 120 min the retina was teased away from the underlying choroid and cut into small pieces. A lo-min rinse with the incubation medium followed the glutaraldehyde fixation. Then, generally, the pieces of tissue were transferred to the osmium tetroxide fixative,
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also with an appropriately adjusted tonicity. This fixation was continued at room temperature for 60 min, and was followed by an additional wash in the original incubation medium. The tissue samples were then divided at random into two groups, one for embedding in the glutaraldehyde-urea resin as described below, and the other for Araldite, following a conventional alcoholic dehydration, and the use of propylene oxide as a transitional solvent. Some tissue samples were embedded without the osmium tetroxide postfixation but then, as will be seen, the membranes retained osmotic capabilities for at least a part of the infiltration process. -4s a prelude to embedding in glutaraldehyde-urea, the concentration of the glutaraldehyde was increased stepwise. First, the tissue was transferred into 5% glutaraldehyde in 0.05 M Na-phosphate buffer, pH 7.0. After 15 min the glutaraldehyde concentration was increased slowly by adding successive quantities of 50% glutaraldehyde with its pH adjusted to 4.1. In one set of experiments with hypertonic manipulation, 1% NaCl was added to the glutaraldehyde. The exchange into 50% glutaraldehyde was completed within 30 min. At this stage the tissue was transferred into a glutaraldehyde-urea mixture preparatory to polymerization. The glutaraldehyde-urea embedding procedure has been described elsewhere (Pease and Peterson, 1972). Basically, it consists of preparing in advance a very strong solution of urea dissolved in a minimum quantity of 50% glutaraldehyde, with a carefully adjusted acidic pH. This mixture is stable for a few days at room temperature. Just before use it is diluted with an appropriate additional amount of 50% glutaraldehyde, also with an adjusted pH. At room temperature the mixture begins polymerizing in less than 30 min at a pH of about 4.3. In so far as longer infiltration times seem desirable, tissue is moved through successive changes of freshly blended mixtures. For the present series of experiments, two parts of the stable glutaraldehyde-urea solution were blended with one additional part of glutaraldehyde. Infiltration involved two changes of freshly prepared 2 : 1 mixtures at 15-min intervals, after which the tissue blocks were isolated in small drops of the resin as the mixture began to thicken. The blocks were left to cure at room temperature over a two-night period. Finally, the blocks were mounted with white carpenter’s glue on supports consisting of short lengths of appropriately thick dowel-rod, with tips shaped in a pencil sharpener as truncated cones. Glass knives were used for sectioning, at substantially higher speeds than are conventional. Generally, and unless otherwise noted, sections were stained first with uranyl acetate, and then with lead citrate. However, in order to visualize membranes with positive contrast when postfixation with osrnium tetroxide had not been employed preceding a glutaraldehyde-urea embedment, such sections were exposed to 0~0, vapors. This then COJIstit,uted their only “stain”. 3. Results Pilot experiments indicated that a secondary fixation with osmium tetroxide was required if disc dilatations induced by hypotonic solutions were to be preserved throughout a glutaraldehyde-urea embedment. Otherwise, if only a glutaraldehyde fixation was used, the membranes retained osmotic properties, and swollen discs returned to normal configurations as the glutaraldehyde (or glutaraldehyde-urea) content was rai.sed preparatory to the embedment. A comparison of the first two figures will make this evident, for (aside from staining) both preparations experienced identical treatment excepting only that the tissue of Fig. 2 was postfixed with OsO,, whereas that of Fig. 1 was not. Figure 2 demonstrates the substantial disc swelling and extreme mitochondrial enlargement that might logically be anticipated as a consequence of . . hypotonm mcubation. The swelling was substantially consistent in a given outer segment, and from one preparation to another. However, the swellings are not seen
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at all in the tissue illustrated in Fig. 1, even though these organelles ntlist ~urel! have been similarly enlarged at one time, and to have been swollen even during t8hcA interval of their glutaraldehyde fixation. Evidently all traces of the enlargement,< disappeared, presumably as thematerial, was infiltrated subsequently, with increasing concentrations of glutaraldehyde (or glutaraldehyde-urea mixture) which inevitabl! eventually led to hypertonic conditions being established. Heller: et al. (1971) also have indicated that disc swelling can be induced or reversed after glutaraldehydc fixation by changing the osmolarity of postfixation media. Retinae which were treated with solutiolis in the iso-osmotic range, were postfixed
FIG. 1. Retina incubated in a decidedly hypotonic solution, and then fixed with hypotonic glutsraldehyde. Subsequently, the retina wa8 exposed to hypertonic conditions a8 a prelude to embedding in polymerized glutaraldehyde-urea. The section ultimately was stained by exposure to 0~0, vapor. Sotr normal looking mitochondria a8 well as highly ordered discs. ( x 34,340)
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with 0~0, and were then embedded in polymerized glutaraldehyde-urea, demonstrated fairly well-organized arrays of discs (Fig. 3). Intradisc spaces always were evident. The width of these spaces was somewhat variable, presumably reflecting small osmotic inequalities. The minimum width approached 20 A and was an ordered feature of many areas. Wider intradisc spaces lacked order and specific dimensions, but generally did not exceed 30 .& in width. Individual nienibranes clearly showed a trilitminar pattern. Retinae exposed to decidedly hypertonic solutions which subsequently were embedded in polymerized glutaraldehyde-urea invariably demonstrated very welldefined and uniformly wide intradisc spaces. It made no essential difference whet,her
Pm. 3. Retina treated exactly 080, solution before embedding Note greatly dilated mitochondria,
as in Fig. 1, except in glutaraldehyde-urea. as well a8 swollen
that it was secondarily fixed with a hypotonic Stained with uranyl acetate and lead citrate. and disordered discs. ( x 39,000
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the original exposure was to hypertonic NaCl (Fig. 4) or sucrose (Fig. 6). The. witkh of these spaces approximated 15-20 A, although the gap seemed slightly (- 5 A) wider after NaCl incubation (Fig. 4) than after sucrose (Fig. 6). We believe t,hat this demonstration of an intradisc compartment even under conditions of extreme hypertonicity is a reflection of our improved embedding technique. These same preparations also demonkrated a kilaminar organization of individual
FIG. 3. Retina first incubated, and then sequentially fixed with glutaraldehyde and in isotonic solutions. The retina was embedded in polymerized glutaraldehyde-urea, stained with uranyl acetate and lead citrate. ( x 160,000) FIG. 4. Retina incubated, and then fixed sequentially with glutaraldehyde and 080, hypertonic with N&I. The retina was embedded in polymerized glutarsldehyde-urea stained with uranyl acetate and lead citrate. ( ,-. 160,000) FIQ. 5. Retina treated exactly as for Fig. 4, except t,hat it w&s conventionally embedded in Araldite. The section was stained with uranyl acetate and lead citrate.
osmium and
tetroxide, the section
in solutions and the dehydrated ( ‘i 160,000)
made section and
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membranes. This was particularly evident when the hypertonicity had been established by using NaCl (Fig. 4). When sucrose was used to create hypertonic solutions, the trila,minar patterns then were less evident (Fig. 6).
FIG. 6. The retina was first incubated and then sequentially fixed with glutsraldehyde and 0~0, in solutions made hypertonic with sucrose. It was then embedded in polymerized glutaraldehyde-urea. The section was stained with uranyl acetate and lead citrate. ( x 240,000) I’m. 7. Retina treated exactly as for Fig. 6, except t.hat it was conventionally dehydrated and embedded in Araldite. The section was stained with many1 acetate and lead citrate. ( ~240,000)
The apparent width of the disc membranes demonstrated some dependence on the means used to create hypertonicity. After exposing retinae to hyperosmotic NaCl, and embedding in polymerized glutaraldehyde-urea, the total disc width was about 210 8. When sucrose was used instead of NaCl, the corresponding width was only
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about 165 A. Thus, in subtle ways, the pattern of preservation of membrane structure was influenced by the character of the vehicle used with the fixative. When conventional techniques of dehydration and embedding in Araldite were employed, significant detail was lost. This is evident in Fig. 5 where the intradisc space was not discernible as a separate entity after incubation with hypertonic NaCI. Instead, there was then the appearance of fused membranes so that the full width of each disc appeared to have a pentalaminar pattern of organization such as described previously by Moody and Robertson (1960) Fernandez-Moran (1961), Nilsson (1965), and by Dunn and Adomian (1971). Th us, under these circumstances, it was not generally possible to differentiate what may be called the “inner leaflets” of the disc
FIG. 8. Retina first incubated and then fixed only with glutaraldehyde with NaCl. The glutaraldehyde-urea embedding mixture also contained stained by exposure to 0~0, vapor. (X 118,000)
in a solution made hypertonic 1 “/f NaCI. The section was finally
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membranes from the disc spaces. Furthermore, the “outer leaflets” were not well preserved and differentiated either. After hypertonic sucrose incubation even less structural pattern was retained through conventional dehydration and embedding in Araldite (Fig. 7). A question remains as to whether or not postfixation with osmium tetroxide is rea.lly essential for the demonstration of the intradisc spaces under hyperosmotic conditions. To investigate the possibility that the appearance might be artifactually dependent on osmium tetroxide, retinae exposed to hypertonic media were fixed only in .hypertonic glutaraldehyde, and then embedded in polymerized glutaraldehydeurea without 0~0, postflxation. In some experiments, 1% NaCl was even added to the embedding mixture to insure the maintenance of a high salt concentration. Thin sections of such material are known to retain their membrane lipids which then can be stained with osmium tetroxide vapors (Pease and Peterson, 1972; Peterson and Pease, 1972). The results of these preparative procedures are shown in Fig. 8. The intradisc space is clearly evident, and thus its persistence and demonstration are independent of an osmium tetroxide fixation. Furthermore, it should be noted that there was no essential difference evident in disc morphology between this material, prepared under hyperosmotic conditions, and that illustrated in Fig. 1 which originally was exposed to a hypotonic medium. It is also seen in Figs 1 and 8 that when 0~0, was not used as a fixative, but rather as a stain, the disc membranes looked thinner tha,n otherwise, and were without evidence of trilaminar patterns. Correspondingly, the disc spaces seemed wider than after postfixation and conventional staining. Thus, it would seem that 0~0, vapors stain somewhat different membrane components tha,n do uranyl acetate and lead citrate. This agrees with findings relative to myelin (Peterson and Pease, 1972) and chloroplast membranes (Nir and Pease, 1973), and confirms what Godfrey (1972, 1973) has seen and measured in rod disc membranes. 4. Discussion The most important part of our experiments involved the osmotic manipulation of rod outer segment discs to see whether under any circumstances the intradisc spaces could be obliterated if the preservation technique was adequate. Since these spa,ces were found always to persist after a glutaraldehyde-urea embedment, it was also deemed necessary to compare results with standard preparative techniques to understand the previous failures to observe these spaces that have been recorded in the literature (Moody and Robertson, 1960; Fernandez-Moran, 1961; Nilsson, 1965; and Dunn and Adomian, 1971). We conclude that conventional procedures do not adequately preserve intradisc spaces and their contents. Godfrey (1972, 1973) has already indicated that the contents of the intradisc spaces are stained with acidic silicotungstic acid which has led him to propose that carbohydrate moieties are to be found there. The spaces and their staining characteristics are comparable to relationships we have established in studying the intraperiod complex of myelin (Peterson and Pease, 1972) and “glycocalyx” in other situations (Pease and Peterson, 1972). Thus, there is comparative evidence that the intradisc spaces may represent volumes occupied in life by hydrated carbohydrate gels. Of course, considering that rod discs originate by invagination of the plasma membrane, it is reasonable to consider homologizing their contents with surface coats. A great advantage of the glutaraldehyde-urea embedment is that polymerization occurs, and spatial relationships become fixed, while there is still substantial water
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in the medium. Thus, highly hydrated gels need not be collapsed as probably often occurs in the final stages of conventional dehydration, as the last of the water is removed (Pease and Peterson, 1972). Gel collapse could most plausibly explain the obliteration of the disc cavities and the appearance of pentalaminar patterns in outer segments prepared by usual procedures (Moody and Robertson, 1960; Fern&ndezMoran, 1961; Nilsson, 1965; and Dunn and Adomian, 1971). Measurementsthat have beenderived from X-ray diffraction studiesof fresh retinae have suggestedthat 5-25 A-wide intradisc compartments exist in life (Blaurock and Wilkins, 1969; Worthington, 1971). The larger figure agreesreasonably well with our own finding that the minimal spacesthat survive exposure to undoubted hypertonic solutions are about 15-20 A wide. When we attempted to approximate isotonic conditions, in many places the compartments were no wider than this. Thus, the evidence suggeststhat under normal and stable physiological conditions, this minimal value may pertain. Little or no osmotic swelling need be anticipated in life. Disc swelling in a hypotonic medium is, of course, a well-known phenomenon (Heller, et al. 1971) even though Cohen (1971) has argued that interpretation may be complex. Our own experiments demonstrate a close parallelism in what is seenin outer segment discs and adjacent inner segment mitochondria. There is no question but that the latter are osmometers. Thus, the results emphasize a morphological pitfall: that an aldehyde fixative must not be relied upon to preserve osmotically-dependent spatial relationships. In essence,at least someof the semipermeable properties and characteristics of disc and mitochondrial membranessurvive moderate concentrations of glutaraldehyde. Membrane-bound volumes can soil1be osmotically manipulated. On the other hand, full permeability apparently is achieved by 0~0, (post-) fixation, which thus provides a convenient way of stabilizing spatial relationships that are osmotically dependent. Although less explicitly stated, this was also one of the conclusions of Heller, et al. (1971). ACKNOWLEDGMENTS
This investigation was supported by U.S. Public Health Service Grant NS 284. REFERENCES Blaurock, A. E. and Wilkins, M. H. F. (1969). Nature (London) 223, 906. Cohen, A. I. (1968). J. Cell Bid. 37,424. Cohen, A. I. (1970). Vision Res. 10,445. Cohen, A. I. (1971). J. Cell Biol. 48, 547. Dunn, R. F. and Adomian, G. E. (1971). I n 29th Proc. Electron Microscopy Sot. Amer., p. 268. (Ed. Arceneaux, C. J.) Claitor’s Pub. Co., Baton Rouge, La. Fernandez-Moran, H. (1961). In The Structure of the Eye, p. 521. (Ed. Smelzer, G. K.) Academic Press, New York and London. Godfrey, A. J. (1972). In 30th Ann. Proc. Electron Microscopy Sot. Amer., p. 50. (Ed. Areeneaux, C. J.) Claitor’s Pub. Co., Baton Rouge, La. Godfrey, A. J. (1973). J. Ultra&u& Res. (In press). Heller, J., Ostwald, T. J. and Bok, D. (1971). J. Cell Biol. 48, 633. Moody, M. F. and Robertson, J. D. (1960). J. Biophys. B&hem. Cytol. 7, 87. Nilsson, S. E. G. (1965). J. Ultrastruct. Res. 12, 207. Nir, I. and Pease, D. C. (1973). J. Ultrastruct. Res. (submitted). Pease, D. C. and Peterson, R. G. (1972). J. Ultrastruct. Res. 41, 133. Peterson, R. G. and Pease, D. C. (1970). In 28th Ann. Proc. Electron Microscopy Sot. Amer., p. 334. (Ed. Arceneaux, C. J.) Cla,itor’s Pub. Co., Baton Rouge, La. Peterson, R. G. and Pease, D. C. (1972). J. Ultrastruct. Res. 41, 115. Worthington, C. R. (1971). Fed. Proc. 30,57.