An electron-microscope study of the cytoplasmic inclusions in the ...

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An electron-microscope study of the cytoplasmic inclusions in the neurones of Locusta migratoria By DOREEN E. ASHHURST and J. A. CHAPMAN (From the Department of Zoology and the Rheumatism Research Centre, University of Manchester. Miss Ashhurst's present address is Department of Entomology, University of Minnesota, St. Paul, Minnesota, U.S.A.) With 3 plates (figs, i to 3)

Summary The cytoplasmic inclusions of the neurones of adult Locusta migratoria have been examined in the electron microscope. The mitochondria are easily recognized by their cristae and outer double membranes. Electron-dense inclusions, also with an outer double membrane but possessing numerous closely spaced internal lamellae in various orientations, are probably small lipochondria. Larger and more diffuse inclusions comprising crescent-shaped aggregates of loosely packed parallel lamellae and vesicles are present; the possible significance of these larger inclusions is discussed. A system of numerous small vesicles distributed throughout the cytoplasm makes up the endoplasmic reticulum.

Introduction T H E neurones of insects have been studied extensively by both light and electron microscopy (see, for example, Gresson, Threadgold, and Stinson, 1956; Nath, 1957; Hess, 1958; and other work quoted below). Numerous cytoplasmic inclusions, lipochondria, mitochondria, neurofibrils, and Golgi bodies (dictyosomes) have been described, but the mitochondria are the only cytoplasmic inclusions about which there has been no dispute. The Golgi bodies have been the most controversial inclusions, since it has been suggested that they are artifacts produced by the osmium and silver impregnation techniques used in making Golgi preparations for the light microscope. The locust neurones were chosen for this electron-microscope study as they have been studied both cytologically and histochemically in the light microscope (Shafiq, 1953; Shafiq and Casselman, 1954). An attempt is made in the discussion to correlate the inclusions seen in the electron micrographs with those described in the light microscope.

Methods The locusts were all adult Locusta migratoria, supplied by the Anti-Locust Research Centre, London. The metathoracic ganglion was removed and placed immediately in fixative at 40 C. Two fixatives were used: (a) 1% osmium tetroxide in veronal-acetate buffer, pH 7-4, with the addition of 0-02% of calcium and magnesium chlorides (Palade, 1956; Geren and Schmitt, 1954). This fixative will be referred to as the 'osmium fixative'; [Quarterly Journal of Microscopical Science, Vol. 103, part 2, pp. 147-53, June 1962.]

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(b) the fixative containing 1% calcium chloride recommended for lipid globules by Chou and Meek (1958). This will be described as the 'calcium-osmium fixative'. Fixation was carried out for either 1 or 2 h at 40 C. The ganglia were taken directly from the fixative into 70% alcohol and then dehydrated through graded alcohols and embedded in araldite (Glauert and Glauert, 1958). Some ganglia from the osmium fixative were 'stained' for 1 h in 1 % phosphotungstic acid in 70% alcohol before embedding. Thin sections of the araldite blocks were cut on a Huxley microtome and were examined in a Siemens Elmiskop I electron microscope at magnifications ranging from 2,500 to 40,000, with double condenser illumination.

Results The neurones are large cells situated in the ganglia between the central mass of nerve-fibres and the connective-tissue sheath. They are surrounded by glial cells. The outer cell membrane, which is double, is very irregular; in some regions it is indented to form a trophospongium (fig. 1). This enables portions of the glial cytoplasm to be brought into intimate contact with the inner portions of the neurone cytoplasm. The nuclei are large and spherical; the nuclear membrane is double. No nuclear pores have been seen. Numerous inclusions occur in the cytoplasm of the neurones. The mitochondria (fig. 3, B) are easily recognized as they are of a typical structure, with round or elongated profiles, an outer double membrane, and a system of irregularly distributed internal cristae. Some inclusions, which occur less frequently, are electron-dense and 0-5 to i-o/i in diameter. They are more or less round in section in cells fixed in calcium-osmium (fig. 2, A), but they have a tendency to be distorted when the osmium fixative is used (fig. 2, B). These inclusions are surrounded by a double membrane and the interior usually contains one or sometimes two lighter (i.e. less electron-dense) homogeneous regions with a diffuse boundary. The electron-dense region comprising the bulk of the interior is mostly granular, but in some parts a system of parallel lamellae is prominent. These lamellae occur in various orientations with respect to the bounding double membrane and both concentric and radial arrangements are found. The periodic spacing of the lamellae is approximately 5 m/x. Usually not more than one or two of these inclusions with closely packed lamellae occurred in a thin section of a single neurone. It will be shown that there is reason to believe that these electron-dense inclusions with tightly packed lamellae are lipochondria. Another type of inclusion, larger and more diffuse than those just described FIG. 1 (plate). An electron micrograph of a neurone of Locusta migratoria at low magnification, showing the nucleus (n), the trophospongium (t), and glial cells (g). Numerous inclusions, most of them mitochondria, occur in the cytoplasm of the neurone. Osmium fixation, araldite embedding.

FIG. I

D. E. ASHHURST and J. A. CHAPMAN

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and occurring with greater frequency, is prominent in the cytoplasm of the neurones after both fixatives. Inclusions of this larger, diffuse type consist of systems of loosely packed, parallel lamellae with vesicles at their ends and scattered around them. These inclusions resemble the Golgi bodies found in other cells, although they tend to be larger. In many instances the lamellae are crescent- or horseshoe-shaped and curve round to form a more or less complete circle or oval (fig. 3, A, B). In some sections an electron-dense body containing small particles and surrounded by a limiting membrane is associated with these crescentic structures. One of these electron-dense bodies is seen in fig. 3, A and another in fig. 3, B, at b. The whole complex, including the crescent-shaped lamellae, is about 1*5 to 2-0/x across. The possible significance of these structures will be considered in the discussion. The endoplasmic reticulum is represented only by small vesicles associated with a large number of dense particles, presumably ribonucleo-protein; it is known that the cytoplasm is strongly basiphil (Shafiq and Casselman, 1954). There are also some vesicles, surrounded by a double membrane which encloses a number of small vesicles. The nature of these is not known.

Discussion In a cytological study with the light microscope, Shafiq (1953) found that the only cytoplasmic inclusions in the neurones of Locusta are the mitochondria and lipochondria. He investigated the dictyosomes seen after the Golgi techniques and gave evidence that these are produced by the deposition of osmium or silver on the surface of the lipochondria. Histochemical studies showed that the lipochondria are composed of phospholipids and cerebrosides (Shafiq and Casselman, 1954). In electron micrographs the mitochondria are easily distinguished by their outer double membranes and internal cristae, but the lipochondria cannot be identified with such ease or certainty. Since the lipochondria contain phospholipids, it would seem appropriate to mention some facts known about phospholipid globules before discussing the results presented in this paper. In 1939 Schmidt, from his studies on phospholipids with polarized light, suggested that phospholipids tend, in aqueous medium, to form bimolecular layers which alternate with layers of water molecules. He showed that, in droplets, these layers would be concentric. It has since been found that extracts of phospholipids which have been fixed in osmium tetroxide, embedded, and sectioned, appear in electron micrographs as a series of alternating dark and light lines with a periodicity of approximately 4 m/x (Stoeckenius, FIG. 2 (plate). Electron micrographs of the small, dense inclusions in locust neurones. These inclusions exhibit numerous internal closely packed parallel lamellae (I), granular regions (g), and one or more homogeneous regions of lower electron optical density (r). A limiting double membrane (dm) surrounds the inclusions. A is fixed in the calcium-osmium fixative. B isfixedin the osmiumfixativeand shows evidence of preparational damage. Stained with phosphotungstic acid.

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1959). It is interesting to note that whenever the acid haematein test indicates the presence of phospholipid there is a tendency for parallel lamellae to appear in electron micrographs (J. R. Baker, personal communication). In the neurones of Helix aspersa, Chou (19576) found that certain of the lipochondria are made up solely of phospholipids. Later, in an electron-microscope study of these neurones, Chou and Meek (195 8) were able to demonstrate the presence of globules with concentric lamellae having the same position in the cell as the phospholipid globules seen in the light microscope. This structure was only apparent, however, if the osmium fixative contained 1 % calcium chloride; it is thought that calcium ions stabilize the bonding of the phospholipid-water interfaces (Baker, 1958). In preparations fixed in osmium tetroxide containing no calcium ions, the structures present in this region are crescentic and rod-like, composed of lamellae similar in dimensions to those in the globules, and numerous associated vesicles. These are very similar to the Golgi bodies identified in many tissues. But since they have been shown to be produced by fixation in the snail neurones, it is possible that a similar situation may occur in other cells. In identifying lipochondria in the electron micrographs it is necessary to consider size as well as chemical composition. The diameters of lipochondria observed in the light microscope range from 0-4 to 2-6 /u.. In the present study the electron-dense inclusions, 0-5 to I-O/A in diameter and possessing closely packed internal lamellae, correspond closely to the fine structure expected for a globule containing phospholipids. It would therefore seem reasonable to identify these smaller inclusions as lipochondria. Their largest size, however, is 1 fx, while the largest size of the lipochondria seen in the light microscope is 2-6/n; thus the larger lipochondria cannot be accounted for, unless considerable shrinkage has occurred during tissue preparation. The other obvious cytoplasmic inclusions seen in electron micrographs are the crescent-shaped aggregations of loosely packed lamellae and vesicles. These have a marked similarity to the Golgi bodies described in other cells. These aggregations are about 1-5 to 2-0 p in diameter, and they should be readily visible in the light microscope. Their significance must now be considered. It has been shown that the dictyosomes in these cells are formed by the deposition of osmium or silver on lipochondria (Shafiq, 1953); if this evidence is accepted, it excludes the possibility that these crescentic aggregations are Golgi bodies. Their larger size suggests that they may arise from the larger lipochondria. It is conceivable that the crescentic appearance might be due FIG. 3 (plate). Electron micrographs of larger, diffuse inclusions in locust neurones. A is a section of part of a neurone fixed in calcium-osmium. It shows a crescent-shaped inclusion with circumferentially arranged lamellae (c) associated with numbers of vesicles (v). A dense, membrane-limited body containing granules (6) occurs at the centre of the inclusion. B is also fixed in calcium-osmium and shows an inclusion with circumferentially arranged lamellae and vesicles arranged in an almost complete circle. The granules and limiting membrane of the central body (6) are clearly visible. Several mitochondria (m) are also present in the field of view of this section.

FIG. 3 D. E. ASHHURST and J. A. CHAPMAN

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to disruption or disorientation of a spheroidal body, incompletely fixed owing to its size; the curvature of these aggregations is such as to suggest that the ends might have been joined in the living cell and that a break occurred during fixation or dehydration. This would imply that the electron-microscopic appearance of the lipochondria is determined by their size—i.e. the smaller lipochondria would be fixed so that they retained their shape, whereas fixation of the larger ones caused them to split open. Chou and Meek (1958) have suggested that the crescentic bodies are an artifact produced by the action of the osmium fixative on the lipochondria; they found that the spheroidal shape was only retained after calcium-osmium fixation. This is in contrast to the findings of the present study in which the crescentic bodies were found with both fixatives. A recent electron-microscope study by Malhotra and Meek (1961) of cytoplasmic inclusions of the neurones of the prawn, Leander serratus, also indicates that the horseshoe appearance may arise by distortion during preparation of the specimen. Spherical bodies, staining in life with neutral red and containing phospholipids, appear as aggregations of membranes in a rodor crescent-shaped form after fixation in Palade's osmium fixative. After the addition of CaCl2, BaCl2, or sea-water to the fixative, some of the inclusions are oval or circular, although still made up of loosely packed lamellae. Neither the smaller dense inclusions noted in the present investigation nor inclusions such as those found by Chou and Meek (1958) after calcium-osmium fixation are described by these authors. The electron-microscopic evidence presented in this paper does not altogether accord with the view that the smaller inclusions and the larger crescentic aggregations are derived from the same type of cell organelle. The smaller electron-dense inclusions are surrounded by a conspicuous double membrane, but no such membrane surrounds any part of the crescentic aggregations of lamellae and vesicles; if these larger aggregations had been formed by disruption one might expect to see remnants of this limiting membrane. Moreover nothing corresponding to the lighter homogeneous regions of the smaller inclusions occurs in the larger crescentic aggregations; the central structure in these aggregations is granular in appearance and is limited by a distinct membrane. It is noteworthy that there are no intermediate forms between the smaller dense inclusions and the larger and more diffuse aggregations. It may also be significant that Shafiq (1953) found minor differences in the staining reactions of the larger and the smaller spheroidal inclusions. These observations suggest that a fundamental difference may exist between the larger and the smaller lipochondria. It is also conceivable that the crescentic aggregations are the product of the diffuse phospholipids in the cytoplasm; fixation might cause these to become orientated into layers of lamellae and vesicles. Dense globules with concentrically arranged lamellae have been described in other cells, both vertebrate and invertebrate (Clarke, 1957; Karrer, i960), but only a few authors have suggested that they may represent phospholipid

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globules (Campiche, i960; Chou and Meek, 1958; Porter and Yamada, i960). There is no evidence to exclude the possibility that the molecules of other substances are orientated in such a way that they would give a similar appearance in micrographs. Golgi bodies have been described in most cells, but as information on the nature of the Golgi inclusions seen in the light microscope increases, the correspondence of the light and electron microscope bodies becomes less certain. Recently Malhotra (1959, i960) has shown that the Golgi network seen in mammalian and bird neurones is formed as a result of the reduction of the osmium tetroxide or silver salts in the basiphil region of the cytoplasm, which incorporates the Nissl substance; this reduction is thought to be performed by the membrane component present. In invertebrate neurones lipids are known to be responsible for the reduction (Shafiq, 1953; Chou, 1957a). Further, Malhotra and Meek (i960) found that the region corresponding to the Golgi network in owl neurones was occupied by the endoplasmic reticulum; Golgi bodies, i.e. aggregations of lamellae and vesicles, were very rarely encountered in the micrographs of the owl cells. The possibility that diffuse cytoplasmic lipids might be responsible for the Golgi bodies seen in the electron microscope has not been examined. Bradbury and Meek (1958) suggest that the membranes in a region of the cytoplasm of the adipose cell of Glossiphonia known to contain phospholipids may be composed of phospholipid-protein and have a bi-molecular structure. But until more cells have been studied both histochemically and in the electron microscope, the problem of the correspondence of the structures seen in the light and electron microscopes will remain. The authors wish to thank Professors H. G. Cannon, F.R.S., R. Dennell, and J. H. Kellgren for their interest and encouragement and Dr. J. R. Baker, F.R.S., for his helpful comments. Grateful acknowledgement is made to the Nuffield Foundation for its generous support. Thanks are also due to Mr. S. Grundy for technical assistance.

References BAKER, J. R., 1958. J. Histochem. Cytochem., 6, 303. BRADBURY, S., and MEEK, G. A., 1958. J. biophys. biochem. Cytol., 4, 603. CAMPICHE, M., i960. J. Ult. Res., 3, 302. CHOU, J. T. Y., 1957a. Quart. J. micr. Sci., 98, 47. 1957*- Ibid., 98, 59. and MEEK, G. A., 1958. Ibid., 99, 279. CLARKE, S. L., 1957. J. biophys. biochem. Cytol., 3, 349. GEREN, B. B., and SCHMITT, F. O., 1954. Proc. Nat. Acad. Sci. Wash., 40, 863. GLAUERT, A. M., and GLAUERT, R. H., 1958. J. biophys. biochem. Cytol., 4, 191. GRESSON, R. A. R., THREADGOLD, L. T., and STINSON, N. E., 1956.

HESS, A., 1958. J. biophys. biochem. Cytol., 4, 731. KARRER, H. E., i960. Ibid., 7, 357. MALHOTRA, S. K., 1959. Quart. J. micr. Sci., ioo, 339. i960. Ibid., 101, 69. and MEEK, G. A., i960. Ibid., 101, 389. 1961. J. Roy. micr. Soc, 80, 1.

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the Neurones of Locusta NATH, V., 1957. Nature, 180, 967. PALADE, G. E., 1956. Proc. third intern, conf, electron microscopy, London,

London (Roy. Micr. Soc). PORTER, K. R., and YAMADA, E., i960. J. biophys. biochem. Cytol., 8, 181. SCHMIDT, W. J., 1939. Nova. Acta Leop., 7, 1. SHAFIQ, S. A., 1953. Quart. J. micr. Sci., 94, 319. and CASSELMAN, W. G. B., 1954. Ibid., 95, 315.

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153 1954, p. 129.