Prismatic and Other Unusual Arrays of Mitochondria1 Cristae in Astrocytes of Cats and Hamsters'

RICARDO MORALES AND DONALD DUNCAN Department of Anatomy, The University of Texas Medical branch, Galveston, Texas 77550

ABSTRACT Further work with mitochondria containing prismatic cristae has revealed that this type occurs as a constant feature and in some abundance in the dorsolateral region of the spinal cord of the cat at cervical, thoracic and lumbar levels. All such mitochondria occur in astrocytes. The most common forms are long rods with an abundance of matrix and very few cristae. The matrix in the majority of such mitochondria is of moderate density and without discernible organization. In others the matrix consists of closely packed rodlets with or without occasional prismatic cristae. A frequent pattern is a core of longitudinal rodlets surrounded by a rim of circumferential ones. In the circum- ferential zone a single row of triangular cristae is present. The unusual mito- chondria vary greatly in size, from 0.2-5.0 ,a. Mitochondria containing ordered arrays of cristae that are triangular in cross section are for the most part much smaller than those with very few cristae and smaller than similar mitochondria described in the Syrian hamster. It is concluded that close packing in the matrix (obviously hexagonal in many instances) is the cause of the prismatic shape of the cristae.

Mitochondria with prismatic cristae were first described and illustrated by Revel, Fawcett and Philpott ( '63) . Since then prismatic cristae and various other paracrystalline arrays in mitochondria have been found in many tissues from a wide variety of plants and animals; see Suzuki and Mostofi ('67) and Korman et al. ('70). Mitochondria in astrocytes of the cat exhibiting cristae that are triangular in cross section were illustrated but not commented on by Mugnaini and Walberg ('64). Blinzinger, Rewcastle and Hagar ('65) described such mitochondria in the astrocytes of hamsters and published ex- quisite pictures of their appearance. Simi- lar mitochondria have been reported by Sheridan ('70) to occur in the astrocytes of the hamster pineal body. No other re- ports on prismatic cristae in astrocytes have been found.

Other peculiarities of rat astrocyte mito- chondria were first mentioned by Hartman ('56) and later illustrated by Farquhar and Hartman ('57). The structures they saw were long filamentous rods with double limiting membranes and very occasional

cristae. Similar bodies were noted by Schultz and Pease ('58) and Gray ('59). None of these reports have commented on the highly organized matrix that surrounds cristae that are triangular in cross section nor do they fully describe other remarkable features of astrocyte mitochondria.

The present report calls attention to the paracrystalline matrix of mitochondria with prismatic cristae, to mitochondria with paracrystalline matrix and without cristae, and to the fact that astrocytes of both hamsters and cats contain an array of unusual mitochondria of which those with prismatic cristae are the least com- mon. Their invariable occurrence and rela- tive abundance in certain regions of the cat spinal cord are also recorded.

MATERIALS AND METHODS

Material studied was obtained from eight Syrian hamsters and four cats. All the ani- mals were apparently full grown and healthy. No account was kept of their sex. The hamsters were perfused through the

Received April 9, '71. Accepted June 7, '71. 1 Supported in part by DHEW grant NS-00690.

545 ANAT. REC., 171: 545-558.

546 RICARDO MORALES AND DONALD DUNCAN

heart with 3% glutaraldehyde in 0.1 M Sorenson's phosphate buffer (Sabatini, Bensch and Barrnett, '63). The cats were perfused with a mixture of glutaraldehyde and paraformaldehyde with sodium caco- dylate buffer as described by Bodian ('70). After perfusion thin slices of cerebellar cortex, deep cerebellar nuclei and spinal cord were placed in ice cold buffered fixa- tive for an additional two to four hours. The material was then washed with phos- phate or cacodylate buffer and transferred to 1% phosphate buffered osmium tetrox- ide for one to two hours. Thereafter it was rinsed in 0.85% sodium chloride, rapidly dehydrated with a series of increasing con- centrations of ethanol and embedded in Epon 812 (Luft, '61).

Sections were cut with glass or diamond knives in a Porter-Blum Sorvall microtome. They were stained with lead citrate (Rey- nolds, '63) or uranyl acetate (Watson, '58; followed by lead citrate. The stained sec- tions were studied and photographed in RCA EMU 3D and 3G microscopes.

OBSERVATIONS

Examples of prismatic cristae displaying triangular outlines in cross section were €ound in astrocytes located in the cere- bellum and the spinal cord of both cats and hamsters (figs. 1-4). They appear to be the same as those illustrated in previous reports and the dimensions of the cristae in both species examined are close to identi- cal. However, prior descriptions did not call attention to the precise arrangements in the matrix associated with the triangular profiles as shown in figures 1-4. In these figures the matrix appears as an array of tiny electron opaque beads or dots each surrounded by an electron lucent shell. From center to center these beads are about 150 A apart. Although there are many flaws in the pattern, the dots in general are arranged in rows that are either parallel to one side of a triangular crista or they intersect one side in the middle of its length at an angle that places the row parallel to one other side of the same tri- angle (figs. 2, 4). It is evident also that three rows of dots occupy the same width. as one side of each triangle. Straight lines can be drawn across the rows of beads in three directions outlining equilateral tri-

angles (fig. 4). More often than not the rows are less apparent along one axis than they are along the other two. Straight lines may be drawn between many four adjacent beads and a diamond shaped figure is out- lined. These arrangements suggest packing in the matrix wherein each dot or bead cccupies the center of small hexagon. Ap- paently as a result of hexagonal packing in the matrix, non-rigid circular outlines of considerably greater diameter than the components of the hexagonal pattern are forced to assume a triangular shape. The matrix packing forbids circular shapes and aside from complete collapse, a triangle encloses the least area for a given perim- eter. It should be noted (fig. 1 ) that mito- chondria exhibiting prismatic cristae often have a peripheral zone where the cristae are tubular and the matrix around them lacks the dotted appearance associated with the triangular profiles. Also there are mito- chondria with paracrystalline matrix and very few or no cristae (figs. 5, 6) . Cristae in these mitochondria when present are prismatic (fig. 5) but their presence is not essential for precise order in the matrix

In longitudinal sections (figs. 7, 8) mito- chondria containing prismatic cristae dis- play linear patterns that vary in detail but they are always characterized by very dark parallel lines separated from one another by lines of lesser density. The darker lines can be accounted for as due to arrest of electrons by sides of cristae that are at right angles to the plane of the section. Although other explanations for the longi- tudinal appearances of lines of different densities are possible it was noted that the darker lines are absent in longitudinal sec- tions of mitochondria with ordered matrix and no cristae (figs. 9, 10). In these only fine parallel lines of uniform opacity are present. From the longitudinal appearance it is inferred that the matrix dots seen in cross sections are end on views of parallel fine filaments.

In addition to containing a central core of uniformly dense parallel filaments many mitochondria, such as depicted in figures 9, 10, are characterized by a peripheral single row of prismatic cristae. These cristae are triangular in cross section and they are circumferentially arranged. The matrix in

(fig. 6).

ASTROCYTE MITOCHONDRIA WITH UNUSUAL CRISTAE 547

the vicinity of the triangles has the samc dotted appearance seen in other areas con- taining the prismatic cristae.

Less spectacular but still unusual mito- chondria outnumber the more bizarre forms in hamster astrocytes (fig. 11). In general such mitochondria are character- ized by their being larger than the ever so many more numerous ones with the usual features. All contain tubular cristae and in all the tubules tend to be arranged in parallel arrays and in many the interior of the mitochondrion is mostly matrix. The matrix in this variety of mitochondria shows no sign of paracrystalline ar- rangement.

A more common type of mitochondria than those with angular cristae, particu- larly in hamster astrocytes from the cere- bellum, is one with tubular cristae ar- ranged as a series of parallel sheets or layers (figs. 12-14). Within each layer the tubules are evenly spaced by interven- ing dark matrix while the matrix separat- ing one layer from another is much more electron lucent. When such mitochondria appear swollen, as many of them do, the sheets may become widely separated but little or no separation occurs between the parallel cristae within a single sheet. Mito- chondria of this variety display a variety of patterns when sectioned longitudinally but all of them have one feature in com- mon; namely, a suggestion of helical ar- rangement of the layers along the long axis of the mitochondrion (figs. 13, 14).

In the cat spinal cord the most frequent types of unusual mitochondria in the astro- cytes are those that contain very few cristae (fig. 15). The largest of these bodies are 5 in diameter and of undetermined length, the smallest are about 0.2 in diameter. They bear little resemblance to ordinary mitochondria and would be un- recognizable as such if not for the presence of abundant intermediate forms. The matrix density of these mitochondria varies considerably but they are never clear or vacuolated as might be expected if their size was due to agonal or post-mortem swelling. In the majority of this type the matrix is finely granular and without indi- cation of orderly array. Others are con- structed as shown in figures 9, 10 with a central core of parallel fine filaments and

a peripheral zone of prismatic cristae that are arranged at right angles to the central filaments. Still others contain parallel tubu- lar cristae in variable numbers and ar- rangements and least common are those with prismatic cristae evenly dispersed in the matrix. In the cat those of the last men- tioned type are usually very small and contain only three or four triangular outlines.

The unusual astrocyte mitochondria are most abundant in the dorsolateral region of the spinal cord of the cat especially in strands of fibrous glia crossing and bound- ing the dorsolateral fasciculus. Here they may be seen in any section through this region at all levels while elsewhere in the spinal cord they are encountered only once in awhile.

DISCUSSION Korman et al. ('70) discussing para-

crystalline arrays in mitochondria attri- bute the prismatic cristae in hamster astro- cytes to ordered packing of the matrix without further analysis or explanation. Other accounts have not specifically men- timed the arrangement of the matrix as a factor to be considered in accounting for the shape of the cristae. Although hexa- gonal packing of matrix filaments appears to be a plausible explanation for the tri- angular shape of the prismatic cristae, the authors have been unable to secure or de- velope a mathematical analysis or proof as to why this is so. Work with simple non-living models such as applicator sticks and plastic tubing of appropriate sizes results in the tubing assuming triangular outlines in cross section when compression is applied to such an assembly.

Such experiments suggest that the cristae become triangular due to some simple mechanical principle. However, it should be understood that the forces induc- ing the assumed hexagonal packing in the matrix are believed to be internal and cohesive as opposed to the external force apdied in the non-living models.

While i t is possible that the prismatic pattern is the result of shrinkage induced by fixation, the wide occurrence of the pattern in many species and many dif- ferent tissues in addition to the general good quality of many of the preparations

548 RICARDO MORALES AND DONALD DUNCAN

that display these Eorms speak for their presence in the living state.

Little can be offered in terms of origin and purpose served by mitochondria with prismatic cristae such as herein described. The fact that cristae with prismatic out- lines have been seen in many species and many locations, rules out any unique genetic factor. It is possible that these forms occur in all animals and serve or are associated with some function common to all. The scarcity of such mitochondria may be due to loss of their characteristic shape in the preparatory procedures al- though this seems very doubtful at the present time. Considering the work of Hackenbrock ('66) and of Green and his associates ('69) on nonenergized and energized states and how long these dra- matic changes in morphology were hidden by accepted fixations for electron micros- copy, i t is not too much to hope that ths functional accompaniments of the pris- matic state will be revealed in the future. The fact that mitochondria with para- crystalline arrangements seem to occur as a regular feature in the dorsolateral region of the cat spinal cord should make experi- mental study of the objects feasible.

The other more numerous mitochondria with parallel tubules arranged in sheets suggest themselves as precursor arrange- ments to the prismatic form and probably in turn arise from less ordered arrays. That matrix organization may play a role in all of the unusual forms of astrocyte mito- chondria is suggested by the occurrence of mitochondria that contain an ordered matrix without cristae.

The spiral arrangement of the cristae in all longitudinal sections of mitochondria containing ordered tubular arrays is re- garded as indicative of a twisting of the entire organelle on its long axis. Since it has been known since the earliest days of cinematography of living cells that mito- chondria bend, undulate and move of their own accord, it is not at all surprising that some may twist when they are elongate in form.

LITERATURE CITED Blinzinger, K., N. B. Rewcastle and H. Hager 1965 Observations on prismatic-type mito-

chondria within astrocytes of the Syrian ham- ster brain. J. Cell Biol., 25: 293-303.

Bodian, D. 1970 An electron microscopic char- acterization of classes of synaptic vesicles by means of controlled aldehyde fixation. J. Cell Biol., 44: 115-124.

Farquhar, M. G., and J. F. Hartman 1957 Neuroglial structure and relationships as re- vealed by electron microscopy. J. Neuropath. and Exp. Neurol., 16: 18-39.

Gray, E. G. 1959 Electron microscopy of neuro- glial fibrils of the cerebral cortex. J. Biophysic. and Biochem. Cytol., 6: 121-122.

Green, D. E., D. F. Korman, G. Vanderkovi and E. Valdivia 1969 Structure and function of the mitochondrial system. Symposium on autonomy and biogenesis of mitochondria and chloroplasts. Canberra City, Australia.

Hackenbrock, D. R. 1966 Ultrastructural basiq for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes in metabolic steady state in isolated liver mitochondria. J. Cell Biol., 30: 269-297.

Hartman, J. F. 1956 Electron microscopy of mitochondria in the central nervous system. J. Biophysic. and Biochem. Cytol., 2: No. 4 suppl.,

Korman, E. F., R. A. Harris, C. H. Williams, T. Wakabayashi, D. E. Green and E. Valdivia 1970 Paracrystalline array patterns seen in mitochondria. Bioenergetics. 1 : 387-404.

Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414.

Mugnaini, E., and F. Walberg 1964 111. Ultra- structure of neuroglia. Ergeb. d. Anat. u. Ent- wicklungs., 37: 194-236.

Revel, J. P., D. W. Fawcett and C. W. Philpott 1963 Observations on mitochondrial structure. Angular configurations of the cristae. J. Cell Biol., 16: 187-195.

Reynolds, E. S. 1963 The use of lead citrate at high pH as an electron-opaque stain in elec- tron microscopy. J. Cell Biol., 17: 208-212.

Sabatini, D. D., K. Bensch and R. J. Barrnett 1963 Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19-58.

Sheridan, M. N., and R. J. Reiter 1970 Observa- tions on the pineal system in the hamster. 11. Fine structure of the deep pineal. J. Morph.,

Schultz, R. L., E. A. Maynard and D. C. Peaae 1957 Electron microscopy of neurons and neuroglia of cerebral cortex and corpus callo- sum. Am. J. Anat., 100: 369-407.

Suzuki, T., and F. K. Mostofi 1967 Intramito- chondrial filamentous bodies in the thick limb of Henle of the rat kidney. J. Cell Biol., 33:

Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478.

375-378.

131: 163-177.

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PLATES

All figures S ~ G W mitochondria found in astrocytes.

PLATE 1

EXPLANATION OF FIGURES

1-2

3-4

From an astrocyte in hamster cerebellum.

From an astrocyte in cat spinal cord.

Figs. 1 and 3 The same magnification ( X 80,000) and enlargements of portions of these figures represented by figures 2 and 4 are also at the same magnification ( x 285,000).

It may be noted that the dimensions of the triangular cristae, the size of the matrix dots and their spacing are essentially identical in the two species. Also note in figure 1 that there is a peripheral zone of unorgan- ized matrix and in this location the cristae appear to be tubular. Straight black lines have been added to figure 4 in accordance with the description in the text.

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ASTROCYTE MITOCHONDRIA WITH UNUSUAL CRISTAE Ricardo Morales and Donald Duncan

PLATE 1

55 1

PLATE 2

EXPLANATION O F FIGURES

5 Cross section of a mitochondrion in cat spinal cord. The matrix con- sists of a paracrystalline array of dots (filaments in cross section) and two cristae one of which has a distinct triangular outline and the other which is at the edge of organized matrix has one flat side facing the dotted matrix and a curved line which is not in contact with the dotted matrix. x 113,000.

6 This cross section shows both paracrystalline matrix and unorganized matrix and no cristae. x 250,000.

7 A mitochondria1 profile displaying prismatic cristae in cross and longitudinal section. The image is interpreted as indicating that parallel cristae may be angulated or curved along their long axes. X 56,000.

Longitudinal section from hamster cerebellum. The darker lines are considered to represent sides of triangular cristae that are at right angles to the plane of section and therefore arrest more electrons than the rest of the section. x 110,000.

8

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ASTROCYTE MITOCHONDRIA WITH UNUSUAL CRISTAE Ricardo Morales and Donald Duncan

PLATE 2

553

PLATE 3

EXPLANATION OF FIGURES

9 Longitudinal section of a portion of a mitochondrion from cat spinal cord. The periphery of this mitochondrion is occupied by a single row of triangular cristae surrounded by dotted matrix. The interior of the organelle of which only a small portion is seen, is filled with parallel, longitudinal filaments. x 153,000.

Another example of a mitochondrion with a core of longitudinal fila- ments and peripheral prismatic cristae that apparently encircle tha central filaments. Note that the central mitochondrial filaments are distinctly lesser in caliber than the astrocyte filaments seen at the top of the figure. x 93,000.

A large mitochondrion from hamster cerebellum with tubular cristae some of which are in orderly array and others arranged in a more randcm fashion. There is no sign of precise or paracrystalline struc- ture in the matrix. x 42,000.

12 Cross and slightly oblique sectioning of mitochondrial cristae ar- ranged in layers or sheets of parallel tubules. The matrix is dotted between adjacent crista in any one layer but is less dense and uil- dotted between each sheet cut in cross section. x 64,000.

10

11

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ASTROCYTE MITOCHONDRIA WITH UNUSUAL CRISTAE Ricardo Morales and Donald Duncan

PLATE 3

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PLATE 4

EXPLANATION OF FIGURES

13 A giant mitochondrion from hamster cerebellum containin? tubular cristae arranged in sheets of parallel cristae and showing in the lower left the appearance of a single sheet sectioned longitudinally and in the upper right a number of sheets sectioned longitudinally but at right angles to their width. x 40,000. A mitochondrion from hamster cerebellum that is basically the same as the ones seen in figures 12 and 13 but more clearly displaying spiral or helical twisting of the layers of cristae. x 64,000.

Cross sections of the large mitochondria with moderately dense un- organized matrix and very few tubular cristae that occur in abun- dance in the dorsolateral region of the cat spinal cord. x 25,000.

14

15

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ASTROCYTE MITOCHONDRIA WITH UNUSUAL CRISTAE Ricardo Morales and Donald Duncan

PLATE 4

557