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An unusual central cytoplasmic body in large crayfish neurons: a light and electron microscopic study
TISSUE & CELL 1972 4 (2) 235-252 Published by Longman Group Ltd. Printed in Great Britain
I. NADELHAFT* and J. W. SECHRIST-t
AN U N U S U A L CENTRAL C Y T O P L A S M I C BODY IN LARGE CRAYFISH NEURONS" A LIGHT A N D ELECTRON M I C R O S C O P I C STUDY ABSTRACT. An unusual centrally located cytoplasmic body (~20 micron diameter) sometimes found in large neurons of crayfish abdominal ganglia is described. It is uniquely situated near the terminus of the axoplasmic stream and is surrounded by a lightly staining halo (~ I micron) with some radial striae connecting to the axoplasm. Its composition is primarily membranous, consisting of unbranched tubules and vesicles resembling a type of smooth ER. Scattered islands of polyribosomes, granular Ell, and a few mitochondria are also present. Its location, composition and raetabolic activity (H:Meucine autoradiography) suggest a functional role associated with production of axonal components.
Introduction
THE large n e u r o n s o f crayfish a b d o m i n a l ganglia are characterized by a distinct axoplasmic stream that extends well into the somal cytoplasm. This conspicuous fibrous material sometimes swirls a b o u t the nucleus before dispersing t h r o u g h o u t the cytoplasm (Ross, 1922). In some o f these neurons we have recently observed an unusually large and well defined cytoplasmic structure at the light microscopic level. This structure, a dark staining spherical body ( ~ 2 0 microns in diameter) which may a p p r o a c h the size of the nucleus, occupies a central position in the cell soma. lts position appears to be a focal point of the intracellular axoplasmic stream indicating a possible functional relationship between the two structures. To our knowledge there has been no report in the literature that m e n t i o n s or * VA Hospital (Leech Farm Rd.), Pittsburgh, Pa. 15206, and Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213. t Department of Pharmacology and Anatomy and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213, present address: Department of Anatomy, College of Medicine, University of Arizona, Tucson, Arizona 15213. Manuscript received 24 November 1971.
illustrates a c o m p a r a b l e cytoplasmic structure for either invertebrate or vertebrate neurons. A l t h o u g h the light microscopic cytology of large crayfish neurons has been studied in considerable detail by Ross (1922) and earlier investigators there are very few, if any, published descriptions o f their ultrastructure. Several electron microscopic studies of other crustaceans and a r t h r o p o d s are available (Malhotra and Meek, 1961; Trujillo-Cenoz, 1962; M a y n a r d , 1971), but again there is n o description o f a cytoplasmic b o d y c o m p a r a b l e to that observed in the crayfish. The objective o f this report is to present the results o f light and electron microscopic observations, including preliminary autoradiographic studies, of this p r o m i n e n t cytoplasmic body and its relationship to other cytoplasmic a n d axoplasmic organelles.
M a t e r i a l s and M e t h o d s
Animals
Crayfish (Procambarus clarkii) ranging in size f r o m 3-5 inches were obtained f r o m the L e m b e r g e r Co. in Oshkosh, Wisconsin. They were kept in either cooled tanks (18°C) or at r o o m t e m p e r a t u r e (25"C) for several 235
236 weeks prior to use and were fed a few pellets daily of "Shrimp-el-etts' (Longlife Fish F o o d Products, Harrison, N.J.). The animals selected for use were all in good condition as determined by visual observation. They were anesthetized by exposure to carbon dioxide (about 15 minutes) or by cooling in crushed ice. The abdominal nerve cord was carefully dissected out and processed as described below.
Microscopy Cords were removed, washed in Van Harreveld's ringer (Van Harreveld, 1936) and processed in one of three ways: (a) in Bouin's fluid followed by paraffin embedding, (b) in 5'!; glutaraldehyde in Van Harreveld's Tinger at p H 7 3 for six days with postfixation in I~',; osmium tetroxide in 0.05M phosphate buffer at the same p H for 1 hour followed by dehydration in ascending series of ethanol and embedding in Epon 812, and (c) in 51!; glutaraldehyde in cacodylate buffer (0.067M, p H 7 3 , 6 5 0 milliosmoles) for 3 days followed by postfixation and embedding as in (b) above. Only nerve cords fixed in the latter manner were utilized for electron microscopy. Light microscopic observations were made on both paraffin and epoxy embedded tissue. Several abdominal nerve cords with their six attached ganglia were cut into 8-10 micron paraffin sections and stained with thionin for general observations. The second, third, or fourth ganglion of several additional crayfish cords embedded in Epon were sectioned serially at one micron in the transverse, horizontal, or sagittal planes. These were cut with a diamond knife on a Huxley ultramicrotome and were stained for one minute with 0-5'!,; toluidine blue in 0.5 ~; borax at 60'~C. The epoxy embedded sections, in which the cytoplasmic body was first observed, were used for studying the shape and distribution of the body, for localizing appropriate areas for thin sectioning, and for autoradiographs (see below). Thin sections for electron microscopy were cut on a Porter-Blum MT-1 ultramicrotome with a diamond knife, mounted on F o r m v a r and carbon coated grids, stained for ½ hour with 5 % uranyl acetate, counterstained with lead citrate (Reynolds, 1963) for 2 minutes, and examined in a Phillips--200 electron microscope.
NADELHAFT AND SECHRIST
Autoradiography Crayfish were injected intraganglionically (2rid and 3rd abdominal ganglion; see Fernandez and Davison, 1969) with Ha-L Leucine (5 t~C per ganglion) and were sacrificed 8 days later with the abdominal portion of the nerve cord subsequently processed, embedded in Epon, and cut as described above under Microscopy. Thick Epon sections (one micron) were placed serially on separate water drops on a 1" ", 3" microscope slide and were dried down using a hot plate. These slides were then dipped in liquid enmlsion (Kodak NTB-3 diluted with an equal amount of distilled water) at 45'C, dried, and placed into slide boxes. These were sealed and placed in the refrigerator (6'C) for exposure times of one to three months. Development of exposed slides was done in D-19 developer for 2 min. at 24"C followed by fixing and washing. The dried slides were stained in either toluidine blue or Paragon Multiple Stain (PS 1301), cover slipped, and observed with the light microscope. Observations
Light microscopy A camera lucida drawing (Fig. 1) of a transverse section through the third abdominal crayfish ganglion is included for orientation purposes. Characteristic features of these ganglia are a central neuropil with four giant axons near the dorsal surface and a rind of cell bodies near the ventral surface. In some of the larger cells a prominent spherical cytoplasmic structure can be seen following toluidine blue staining of thick Epon sections of tissue fixed in osmium tetroxide. A typical appearance of this cytoplasmic structure, which we refer to as ~body" for lack of a better term at the present, is shown in Fig. 2. in E p o n sections stained with toluidine blue the body, which is more densely stained than much of the surrounding cytoplasm, is positioned in the center of the neuron and appears to be located near the region where the axoplasmic stream disperses throughout the cytoplasm. In neurons with a diameter of 100 microns it is not unusual for the body to be as large as 20 microns in diameter. It is surrounded by a halo about one micron thick that stains more lightly
CYTOPLASMIC BODY 1N CRAYFISH N E U RO N S than most of the remaining cytoplasm. Light staining strands or striae that are in continuity with a similar staining cytoplasmic network radiate outward from this halo as opposed to the nucleus where the strands can be seen to go around it. This network, in turn, is continuous with the well defined axoplasmic stream, Electron microscopic observations (see subsequent descriptions) reveal that these light staining areas contain fibrillar elements similar to those in the a×oplasm. The appearance of the body from one neuron to another is usually quite similar (Figs. 3-5). Out of approximately 15 examples of such neurons studied in detail thus far in various planes of section only one contains a body which is more oblong than spherical in shape (Fig. 6). In neurons sectioned horizontally, such as the one in Fig. 5, the main axoplasmic stream cannot always be observed because of its position dorsal to the body. However, this plane of section cleaily illustrates the outward radiation of the striae from the body. Other characteristic light microscopic features (Figs. 2 6) of these neurons containing the central cytoplasmic body are numerous crescent-shaped dictyosomes or Golgi bodies, a widely dispersed cytoplasmic substance of intermediate staining density, and a displaced nucleus with one or two prominent nucleoli. Within the intracellular axoplasmic stream thread-like structures which are probably mitochondria can be observed. No Nissl bodies similar to those in large vertebrate neurons are present. However, as will be demonstrated in subsequent electron micrographs, the homogeneous background staining of intermediate density is due to the diffuse distribution of polyribosomes and granular endoplasmic reticulum. The widespread and somewhat isolated distribution of the Golgi bodies in crayfish and other invertebrate neurons also differs from those of vertebrate neurons which often are located in a perinuclear position. It has been suggested that the vertebrate configuration of the Golgi bodies may be partly due to fixation procedures (Shantha, Manocha, Bourne, and Kappers, 1969). The cytoplasmic features of comparable neurons without the body differ little, if at all, from those containing the body. Therefore, there is nothing to indicate D
237
that neurons containing the body are in any way degenerative or pathological. In paraffin embedded sections fixed with either formalin or Bouin's solution instead of osmium tetroxide the surrounding cytoplasm, but not the body, is stained with basic dyes such as thionin or toluidine blue. In at least one instance we located a negatively stained sphere in the central cytoplasmic zone (Figs. 7, 8). Although much of the surrounding cytoplasm is stained, Golgi bodies are not apparent. This indicates that the central body is probably primarily membranous in composition, as are the Golgi bodies, and requires a fixative such as osmium for its preservation.
Topographical distribution Prior to describing the fine structure of the body, a brief account of our current understanding of the geographical distribution of crayfish neurons containing the body is presented. This distribution has been studied by observing serial one micron sections of abdominal ganglia cut in either transverse, sagittal, or horizontal planes. Out of 15 examples studied thus far the body has been found in bilaterally symmetrical neurons in at least two instances. The cells in Figs. 3 and 4 represent one such pair located rostrally in the 3rd ganglion. Such symmetry has not been established as a general property however. For example, after making a series of camera lucida drawings of horizontal sections through the second abdominal ganglion (see Fig. 17), it was determined that out of a total of four neurons containing the body three were located on one side of the midline and only one on the other. A few of the neuron somas containing the cytoplasmic body are located in positions comparable to certain of those which have been mapped for 9 fast flexor motor neurons whose axons exit by way of the third roots (Kennedy, Selverston, and Remler, 1969). In one instance, the axon of such a neuron located in the rostral part of the 3rd ganglion was traced by means of serial sections further rostrally into the nerve cord as it crossed the midline and assumed a dorsolateral position in the nerve cord. Although it was not traced all the way to the third root, this axon probably originated from one of the fast flexor motor neurons innervating the next rostral segment via the third root.
238
NADELHAFT
AND
SECHRIST
Fig. 1. Camera lucida drawing of a transverse section through the 3rd abdominal crayfish ganglion at the level of emergence of the first pair of roots. Typical features illustrated are a rind of cell bodies adjacent to the ventral ('q) surface and medial Imgl and lateral fig) giant axons near the dorsal (D) surface. The central core or neuropil (Np) contains numerous nerve cell processes of which only a few are shown. A spherical "body' is indicated (B, arrows) in each of two laterally placed nerve cells on the left side of the ganglion. Fig. 2. Light micrograph of a large nerve cell from a region comparable to that indicated by arrows in the above drawing (Fig. I ). In the center of this nerve cell is a prominent darkly stained body (B) which is surrounded by a light halo that is comparable in staining intensity to the axoplasmic stream (As) and a light network distributed throughout the cytoplasm. Other intracellular structures shown at this low magnification are the nucleus (N/ and numerous darkly stained oval dictoyosomes (Golgi bodies). [n the field of view are parts of other nerve cells, glial cell nuclei (*), and a portion of the neuropil (Np). Epon section stained with toluidine blue. × 650. Figs. 3, 4. Light micrographs illustrating two examples of the body as it appears in sagittal sections. Again note the position of the body in relation to the axoplasmic stream and the light staining striae radiating from the body. These two neurons occupied bilateral symmetrical positions in the rostral 3rd abdominal ganglion. Epon sections stained with toluidine blue. × 640. Fig. 5. Light micrograph illustrating an example of the body as it usually appears in horizontal sections. In this neuron (from 2nd abdominal ganglion) and the one in Fig. 6 the main axoplasntic stream is dorsal to the plane of section. The light halo with striae radiating out fi'om it is clearly shown. Note the position of the body with respect to the light staining nucleus. Epon section stained with toluidine blue. >:640. Fig. 6. Light micrograph illustrating an example of an unusually shaped body in a horizontal section. [t is more obloag than spherical. Epon section stained with toluidine blue. :: 400. Figs. 7, 8. Light micrographs of two transverse paraffin sections (7 microns thick) through a large crayfish neuron in the 3rd abdominal ganglion. An intervening section is omitted. In Fig. 7 the body appears as a negatively stained sphere in the center of the basophilic cytoplasm; the unstained region to the left of the body is part of the nucleus. In Fig. 8 the section passes through the middle of the nucleus including a prominent nucleolus: clearly shown is the unstained axoplasmic stream penetrating the cytoplasm with some of it dispersing in the general vicinity of the body shown in Fig. 7. Bonin's fixative, thionin stain. × 740. Fig. 9. Electron micrograph of a strip of cytoplasm between the nucleus (N) below and the body (B) above. The cytoplasm is characterized by a relatively uniform distribution of mitochondria (M), Golgi bodies (G), clusters of polyribosomes (R), and endoplasmic reticulum (ER). The ER is primarily the branched tubular variety with some profiles having a few attached ribosomes while others have none. Ribosome clusters lie between the ER profiles. The body contains membranous vesicles and tubules (see Figs. 10-12). It is surrounded by a halo (H) of agranular cytoplasm. Same cell as Fig. 2. ,. 14,750. Fig. 10. Electron micrograph of a strip of body (B) and adjacent cytoplasm (Cyt) including the intervening transitional region or halo (H). The field of view extends from the middle of the body above toward the cytoplasm below that is between the body and the nucleus. Although the body consists primarily of large vesicles and tubular profiles of smooth endoplasmic reticulum, small islands of granular ER, polyribosomes (R), and an occasional mitochondrion (M) with associated glycogen particles can be observed. The rectangular area is shown at higher magnification in Fig. 12. Same cell as Fig. 2. z 14,750.
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242 It s h o u l d be e m p h a s i z e d t h a t the ganglia o f s o m e crayfish d o n o t h a v e n e u r o n s c o n t a i n ing t h e b o d y . H o w e v e r , if n e u r o n s w i t h the b o d y are f o u n d in o n e crayfish g a n g l i o n , t h e y are u s u a l l y f o u n d in o t h e r g a n g l i a o f t h a t s a m e crayfish a n d in o t h e r crayfish f r o m the s a m e g r o u p . T h u s far we h a v e s t u d i e d five different g r o u p s o f crayfish. N e u r o n s cont a i n i n g the b o d y were f o u n d in the a b d o m i n a l g a n g l i a o f three o f these five g r o u p s (see Discussion for additional comments).
Electrotl microscopy C y t o p l a s m i c features, o t h e r t h a n the central b o d y , of large crayfish n e u r o n s are generally c o m p a r a b l e to m a n y o t h e r i n v e r t e b r a t e
n e u r o n s at the u l t r a s t r u c t u r a l level (Figs. 9, 13-15). T h e e n d o p l a s m i c r e t i c u l u m is widely d i s t r i b u t e d t h r o u g h o u t the c y t o p l a s m . It c o n s i s t s p r i m a r i l y o f the b r a n c h e d t u b u l a r variety with an irregular attachment of ribosomes. The percentage of ribosomes a t t a c h e d to these m e m b r a n e s is low. C l u s t e r s o f free p o l y r i b o s o m e s d i s t r i b u t e d b e t w e e n the t u b u l a r E R profiles are n u m e r o u s . I n c o n t r a s t w i t h v e r t e b r a t e n e u r o n s the scarcity o f s t a c k e d cisternae o f g r a n u l a r e n d o p l a s m i c r e t i c u l u m is striking. T h i s results in the diffuse b a s o p h i l i a o r b a c k g r o u n d s t a i n i n g o b s e r v e d at the light m i c r o s c o p i c level with the a b s e n c e o f p a r t i c u l a t e N i s s l bodies. O t h e r c y t o p l a s m i c organelles exhibit n o
Fig. I I. Electron micrograph of a region in the interior of the body selected to illustrate an area containing clusters of polyribosomes (R), mitochondria (M), and granular endoplasmic reticulum (gER). Such areas are scattered among a primary population of vesicles (V) and smooth tubular profiles iT). Lightly stained microtubules (rot, arrows) appear to form a loose meshwork throughout the body. Same cell as Fig. 2. ,42,500. Fig. 12. Electron micrograph of a region in the interior of the body (see rectangle in Fig. 10) with the typical distribution of vesicles (V) and smooth tubular profiles IT). The diameter of those structures appearing as circles in cross section ranges between 300 and 2500~ whereas the tubules range between 500 and 1000 .&, The large circles (probably vesicles) generally have an empty appearance except for sometimes containing one or several smaller vesicles. Occasionally the tubules have bulbous ends (large arrow). Microtubules (nat) about 230 ~. in diameter are present in the background; they also may appear to be continuous with the larger tubular structures (double small arrows/. In the upper right corner some typical branched, smooth endoplasmic reticulum (sER) can be seen. Same cell as Fig. 2. , 42,5(10. Fig. 13. Electron micrograph of a large crayfish neuron whose cytoplasmic body (B) and surrounding halo (HI is in direct continuity with part of the main intracellular axoplasmic stream (As, large bidirectional arrows) as well as smaller branches (small arrows) of it. Note vesicles (V) similar to those in the body within the axoplasmic stream. Adjacent to the axoplasm and the halo surrounding the body are typical cytoplasmic components such as endoplasmic reticulum (ER), polyribosomes, and mitochondria (M). The small dense particle~ in the axoplasm are probably glycogen. , 14,750 Fig. 14. Electron micrograph of a crayfish neuron in which much of the axoplasmic stream (As, large bidirectional arrows) by-passes the body (B) as it disperses throughout the cytoplasm. Large fingers of axoplasm (small arrows), however, are in continuity with the halo (H) surrounding the body. Fibrillar elements shown to be microtubules (rot) at a higher magnification can be seen in the axoplasm and its finger-like branches. Several Golgi bodies (G) as well as endoplasmic reticulum (ER), (polyribosomes (R), and mitochondria (M) are present in the adjacent cytoplasm. / 14,750. Fig. 15. Electron micrograph of transition region between the axoplasmic stream and cytoplasm of a large crayfish neuron without a body. The distribution of cytoplasmic organelles such as endoplasmic reticulum (ER), polyribosomes (R), mitochondria (m), and Golgi bodies (G) with associated vesicles are comparable to neurons with the body. Note the numerous microtubules in the intracellular axoplasmic stream (As, bidirectional arrows). × 24,000. Fig. 16. Electron micrograph of a portion of the intracellular axoplasmic stream (As) as it skirts the nucleus (N). The axoplasm contains vesicles, endoplasmic reticulum (ERL and mitochondria (M) in addition to numerous microtubules. Same cell as Fig, 15 above. × 24,000
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246 particularly unusual features. The Golgi bodies appear in fixed tissue as large crescents of flattened sacs or cisternae with numerous associated vesicles (Figs. 9, 14 and 15). They are extensively distributed in a somewhat isolated fashion throughout the cytoplasm with the exception of the central cytoplasmic body and the intracellular axoplasmic stream. The close orientation of some of the Golgi bodies to the axoplasmic stream (Fig. 14) is such that vesicles can easily be ieleased into it. It may be significant that the Golgi bodies vary in size and that some neurons without a central body contain larger Golgi bodies. The mitochondria are numerous with relatively few cristae which sometimes appear to be disrupted by swelling during the fixation process. Dense particles, probably glycogen, are occasionally associated with them (Fig. 10) Sometimes the glycogen particles can be observed adjacent to membrane bound structures filled with electron dense granular material (Figs. 13, 14). These may represent transformed mitochondria since they are similar in size. Typical microtubules are dispersed among the cytoplasmic structures described above and are especially evident in regions described as a light cytoplasmic network at the light microscope level. The large cytoplasmic body, at low magnification (Fig. 10), appears to consist primarily of membranous elements preserved as round vesicles and unbranched tubular structures which are most likely a type of smooth endoplasmic reticulum. These membranous elements are embedded in a lightly stained cytoplasmic matrix that is continuous with the halo which separates the body from the surrounding cytoplasmic organelles. Within the body a few mitochondria and scattered islands of polyribosomes and granular ER can be observed. The latter structures as well as a loose background meshwork of microtubules are better shown at higher magnification (Fig. 11 ). Closer examination of the membranous elements within the body (Figs. 11, 12) reveals that the tubular structures range in diameter from 500 to 1000 ~ . They exhibit no particular orientation within the body. Without extensive serial sectioning it is difficult to determine the length of these tubules or the extent to which they are continuous or separate interwoven cylindrical structures. The majority, however, do not
N A D E L H A F T AND SECHRIST appear to be the typical branched, interconnected tubules most commonly referred to as smooth endoplasmic reticulum. The content of many of these membrane bound tubules is more electron-dense than the surrounding cytoplasm. Occasionally 230 microtubules appear to be continuous with the larger tubular profiles (Fig. 12), but this may be due to overlap within the section rather than actual continuity. Those membrane bound elements within the body which appear to be circular in section (Figs. 1 I, 12) have diameters in the range of 300 to 2500 ~. Scrutiny of the density of material within these circular profiles reveals that most of the smaller diameter circles have a core which is more electron-dense than the background cytoplasm whereas the content of the larger circles is comparable in density to the background cytoplasm with the exception of an electron dense rim adjacent to the surrounding membrane. It would appear that many of the smaller electron-dense circles represent transversely cut tubules since their diameters and electron density are equivalent. The larger circles, on the other hand, are most likely spheres or vesicles. A few of them contain one or more small vesicles. The outer electron-dense rim within the vesicular membrane may be partly due to the inclusion of a portion of the spherical shell within the section ( ~ 1000 ~ thick). A short calculation indicates that a 1000 A section through the middle of a 2000 A sphere would have its edge broadened to a width of approximately 150 ~. An alternative explanation for the presence of an electrondense rim in the larger vesicles is that these spheres may result from "ballooning" of tubules whose electron dense contents cleave in the middle but adhere to the outer membrane. Sometimes the tubules do have bulbous ends (Fig. 12) which would appear to be isolated spheres or vesicles if cut in the perpendicular plane. The distinct intracellular axoplasmic stream that penetrates deeply into the cell soma is composed of numerous microtubules, mitochondria, smooth ER, and vesicles of various sizes (Figs. 15 and 16). Of particular interest in those cells that contain the body is the apparent relationship of the axoplasmic stream to the body. Either part of the main stream may directly
CYTOPLASMIC BODY IN CRAYFISH N EU RO N S approach the body (Fig. 13) or branches of the stream may be given off to the body (Fig. 14) as it disperses throughout the remainder of the cytoplasm. Some of the vesicles in the axoplasmic stream are comparable to those in the central body as well as those associated with the Golgi bodies. It is clear that the light halo surrounding the body and similar light staining areas observed in the cytoplasm at the light microscope level contain microtubules that are in continuity with those in the axoplasmic stream. Neurofilaments such as those seen in vertebrate axons have not been observed although a detailed study has not been made at high magnification.
A utoradiography The observations reported here are taken from material belonging to an unrelated experiment and are therefore of a fragmentary nature. At the present time available autoradiographs of crayfish neurons containing the central body are limited to an 8 day time interval following intraganglionie injection of Ha-leucine. Since these provide useful preliminary information on the metabolic activity of the body, it was deemed appropriate to present them. The autoradiographs shown in Figs. 17-20 were taken after 30 days exposure time. Alternate sections exposed for 90 days revealed such a dense distribution of silver grains that their usefulness for grain counting over neurons was limited. In general, nearly all nerve cells, including those containing the body, revealed high levels of incorporation of H'Meucine. The density of silver grains over the nuclei was slightly lower than over the cytoplasm while nucleoli were significantly more heavily labelled. Axons in the neuropil and nerve cord which originated from cells in the injected ganglion were labelled, but not those from other ganglia. However, the Schwann cell sheaths of all axons within the vicinity of the injected ganglion were labelled irrespective of the origin of the axons. At first glance the grain density over the cytoplasmic body appeared to be comparable to that of the remaining cytoplasm, but more detailed studies of grain counts revealed the density over the body to be slightly lower than the average over the cytoplasm. For example, counts made on four serial sections through the body gave an average of 204- 7
247
counts over the body and 231 ~5 counts within equivalent areas (3 from each section) of the surrounding cytoplasm. For the above measurements an 18.9 micron diameter circle with an area of 282 square microns was used. Closer examination of certain sections through the body at higher magnification reveals some central spots with relatively high grain density (Fig. 18). These could correspond to those areas that contain islands of polyribosomes and granular ER shown in Figs. 10 and 11. However such regions of high and low grain density within the body can also be due to statistical fluctuations in the number of radioactive nuclei which have decayed. The latter possibility is supported by examination of alternate sections (Figs. 19, 20) which reveal considerable variability in grain distribution. Furthermore, examination of sections exposed for 90 days with much higher grain density reveals less of a differential from one area to another. It would appear, therefore, that eight days after injection of H:Meucine the central cytoplasmic body indicates incorporation of labelled precursor at levels nearly equivalent to the surrounding cytoplasm. This clearly establishes the fact that the body is quite active metabolically. Discussion
Agranular or smooth endoplasmic reticulum has been known for some time to have a widespread occurrence in cell types of very different function (Christensen and Fawcett, 1961). To our knowledge, however, there are no published descriptions of invertebrate or vertebrate neurons that contain an aggregate of smooth ER and vesicles comparable to that which we have described in the large cytoplasmic body of certain crayfish neurons. The axons of most nerve cells contain some smooth ER, but within the cell some various configurations of granular ER as well as Golgi bodies are normally the predominant membranous structures. Since smooth ER is not abundantly found in neurons, descriptions dealing specifically with its distribution and potential functional significance in various other cell types are of particular interest. A comparison of representative electron micrographs of
248
NADELHAFT AND SECHRIST
s m o o t h E R a n d the different functional roles a t t r i b u t e d to it in various cells clearly reveals t h a t generalizations c a n n o t be made f r o m one cell type to another. A few of the proposed roles for s m o o t h E R include detoxification m e c h a n i s m s as well as lipid a n d cholesterol m e t a b o l i s m in liver, lipid t r a n s p o r t in intestinal epithelium, biosynthesis of steroid h o r m o n e s in cells of the testis, ovary, a n d adrenal glands, a n d excitation-contraction coupling in skeletal muscle (Fawcett, 1966). A recent electron microscopic study of oenocytes of an insect led to the suggestion t h a t those cells c o n t a i n i n g densely packed s m o o t h E R could be the source of ecdysone a n d the stimulus for m o l t i n g (Locke, 1969). W h a t e v e r the putative functional role these m e m b r a n o u s elements have in various cells, it has been well d o c u m e n t e d that their degree of d e v e l o p m e n t a n d the presence of associated enzymes m a y change m a r k e d l y d u r i n g physiological or e n v i r o n m e n t a l fluctuations (Siekevitz, 1963; Bjersing, 1967). A l t h o u g h we have n o t been able to d e t e r m i n e the functional significance of the large cytoplasmic body in crayfish neurons, certain features of this p r e d o m i n a n t l y m e m b r a n o u s structure have led us to a hypothesis concerning its role. The position o f the b o d y in the cell near the t e r m i n u s of the axoplasmic stream and its c o m p o s i t i o n suggest a function associated with p r o d u c t i o n of certain axonal constituents. The body is s u r r o u n d e d by a halo of light staining material with similar staining striae r a d i a t i n g outward. Some of these light staining areas clearly merge with the main axoplasmic stream whereas others diffuse t h r o u g h o u t the cell soma. Closer e x a m i n a t i o n
at the ultrastructural level reveals such areas contain microtubules which are apparently c o n t i n u o u s with those in either the m a i n axoplasmic stream or those dispersed t h r o u g h o u t the cytoplasm. Such light staining areas m a y represent pathways whereby the body, cytoplasm, a n d axoplasmic stream are interconnected. The body is c o m p o s e d primarily of m e m b r a n o u s elements consisting of spherical vesicles a n d tubules which resemble a type of u n b r a n c h e d , s m o o t h endoplasmic reticulum in b o t h form a n d dimensions. W i t h i n the central p o r t i o n of the b o d y there are also some, but n o t extensive, a m o u n t s of ribosomal elements. These ribosomes are usually f o u n d in scattered small islands c o n t a i n i n g g r a n u l a r ER, clusters of polyribosomes, and a few m i t o c h o n d r i a . T h e m e m b r a n e s of the granular E R a p p e a r to be c o n t i n u o u s w i t h the s u r r o u n d i n g s m o o t h ER. Microtubules f o r m a loose m e s h w o r k t h r o u g h o u t the body in a d d i t i o n to being present in the halo a r o u n d the body. Some of the vesicles a n d s m o o t h E R observed in the axoplasm are c o m p a r a b l e to those of the outer p o r t i o n of the body. Morphologically it would appear, especially in several instances where large fingers of a x o p l a s m a p p r o a c h the body, t h a t some m e m b r a n o u s elements of the b o d y such as vesicles move into the axoplasmic stream from the body where they are formed. The morphological relationships of various structures within the body are not clear. Occasionally a few of the tubular structures have swollen ends which may represent vesicle formation. F r o m other studies, however, it is k n o w n that tubules of s m o o t h e n d o p l a s m i c reticulum are fragile a n d sensitive to fixation procedures. For example,
Fig. 17. Autoradiograph of a horizontal section through the 2nd abdominal ganglion of a crayfish sacrificed 8 days after intraganglionic injection of 5 ~C of HZ~-leucine. The rostral (R) and caudal (C) ends of the ganglion are indicated. The cell identified by the arrow contains a body which is shown at higher magnification in Fig. 18. Closer examination of the distribution of silver grains at intermediate magnification reveals the cytoplasm to be heavily labelled while the intracellular axoplasm, the body, and nucleoplasm, except for the nucleolus, are labelled at somewhat lower levels. Serial sections of this ganglion indicated that four neurons contained the body: a rostral one (arrow) on the right side and three others (not shown) on the left side including a caudal-lateral one photographed in Fig. 5. Emulsion covered Epon section, 30 day exposure, Paragon Multiple Stain. ~: 115.
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Fig. 18. A u t o r a d i o g r a p h at h i g h e r m a g n i f i c a t i o n o f the n e u r o n with a c y t o p l a s m i c b o d y indicated by an a r r o w in Fig. 17. G r a i n c o u n t s over the d a r k l y stained b o d y are slightly less t h a n in e q u i v a l e n t sized regions in the r e m a i n i n g c y t o p l a s m (see O b s e r v a t i o n s for details). T h e overall d i s t r i b u t i o n o f grains a b o v e b o t h the c y t o p l a s m a n d the b o d y is quite u n i f o r m a l t h o u g h a r e a s with g r e a t e r g r a i n density c a n be observed. ~ 625. Figs. 19, 20. A u t o r a d i o g r a p h s o f serial sections o n either side o f t h a t s h o w n in Fig. 18 to illustrate the variability o f g r a i n d i s t r i b u t i o n over the b o d y f r o m o n e section to a n o t h e r . In Fig. 18 m a n y o f the grains are c o n c e n t r a t e d centrally in a r e a s k n o w n to c o n t a i n r i b o s o m e s a n d g r a n u l a r E R b u t in Figs. 19 a n d 20 n u m e r o u s g r a i n s are also over p e r i p h e r a l p a r t s o f the b o d y . :~:625.
250 in interstitial cells of the guinea pig testis, the tubules of ER are sometimes preserved as vesicles of various sizes if glutaraldehyde is not used as a fixative (Christensen, 1965). Because glutaraldehyde was used routinely in our procedures, the vesicles in the cytoplasmic body of crayfish neurons probably more closely represent the true configuration rather than fixation artifacts although the latter possibility cannot be ruled out. In several instances 230 ~ microtubules have also been demonstrated as appearing continuous with the larger tubular profiles of smooth ER within the body. The latter observation may be significant with respect to the origin of microtubules or transport of materials but we have no evidence for either possibility. The results from autoradiographic studies clearly establish that the central cytoplasmic body is metabolically active. Eight days after injection of H'~-leucine a considerable number of silver glains aie more or less evenly distributed over the entire body. In fact, the level of activity (per unit area) is approximately equal to that of the remainder of the cytoplasm. The question of whether the body is a primary site of synthesis or plays an intermediate role in which it is the recipient of proteins produced elsewhere remains unanswered. The body could be a special packaging station, similar to that of the Golgi apparatus (Droz and Koenig, 1970), where proteins produced in the cytoplasm are modified prior to being transferred to the axoplasmic stream. However, the presence of areas with ribosomes within the body suggests that at least some protein synthesis must occur there as well. Nevertheless the relative paucity of ribosomes compared with the abundance of smooth ER would appear to support the hypothesis of the body serving primarily as an intermediate station for modification or assembly of molecules (structural or secretory) destined for the axon. Further examination of such questions dealing with the initial site of incorporation of labelled precursors, the nature of the final products, and the extent of movement of molecules between the body, cytoplasm, and axoplasm requires additional autoradicgraphic experiments utilizing shorter time intervals between injection of labelled precursor and sacrifice. However, as pointed out below, we aie hampered by the incon-
N A D E L H A F T AND SECHRIST sistent frequency of the body"s occurrence. This difficulty should be resolved before proceeding with additional incorporation studies. The study of functional and metabolic properties of this body under predictable and controlled conditions has been made difficult by our inability to be certain of its occurrence within any particular neuron or any individual crayfish. We have found examples of the body in three out of five different groups of crayfish. Some of the groups with the body were obtained as much as one year apart in experiments carried out in different laboratories. In attempts to differentiate between those groups in which bodies were found and those where they were not found, we have noted the following situations. In two cases where bodies were found the temperature of the water in which the animals were kept was different ( 2 5 C in one case and 18:'C in the other). The abdominal nerve cords were collected following two different kinds of anesthesia, CO2 and cold, and again bodies were found in animals treated by each method. In one group animals in which no bodies were found, the fixation procedure was different from the others in that a lapse of approximately 3½ months occurred between glutaraldehyde fixation and osmium postfixation. These cords showed much less blackening of membranes than did those cords in which osmium followed glutaraldehyde by only a short time. In this group our inability to locate neurons with the body may be due to the fact that membranous structures including Golgi bodies simply could not be clearly recognized. Although we are unable, at present, to predict the circumstances under which the abdominal ganglion neurons will contain these bodies, we have not yet made any extensive investigation of the many possible variables which could be important. Perhaps the presence of the body is related to a cyclic process such as molting with increased metabolic requirements due to growth. Another possibility is a change in the ion composition of the water in which the animals are stored or the length of time during which they are held before use. Also, the type of food used or the amount provided may be significant. Experiments to investigate these variables are being planned prior to further examination of the metabolic
251
CYTOPLASMIC BODY IN CRAYFISH N E U R O N S activity and function of this unusual cytoplasmic body found in some crayfish neurons.
Summary At the light microscope level large neurons of crayfish abdominal ganglia are characterized by dispersed Nissl substance, numerous prominent Golgi bodies, and a distinct intracellular axoplasmic stream. Observations of tissue postfixed in osmium reveal some of these neurons contain a a darkly stained central cytoplasmic body ( ~ 2 0 microns in diameter) that may approach the size of the nucleus. This well defined spherical body is surrounded by a light staining halo with outward radiating striae some of which are in direct continuity with the axoplasmic stream. Electron microscopic studies indicate the body is primarily membranous consisting of a somewhat isolated aggregate of unbranched tubular structures (500 1000~ in diameter) and spherical structures or vesicles (300-2500 ~ in diametert which together resemble a type of smooth ER. The content of the tubular structures is more electron dense than the background cytoplasm. Within the central region of the body among the tubules and vesicles are scattered islands of polyribosomes, a few mitochondria, and some granular ER. Scattered microtubules (230 ~ ) form a loose background meshwork some of these microtubules appear continuous with
those in the surrounding halo as well as those in the striae that approach the main axoplasmic stream. Many of the vesicles and smooth ER in the axoplasm are similar to those on the peripheral margin of the body. Preliminary HMeucine autoradiography reveals incorporation levels within the body to be nearly equivalent to that of the surrounding cytoplasm. The position of the body near the terminus of the axoplasmic stream, its composition, and its inconsistent presence suggest a cyclic functional role associated with production of certain axonal constituents during undetermined physiological or environmental fluctuations.
Acknowledgements We thank Linda Rose and Arnitia Allison for histological assistance and Charles Abbott and Donald Ferrera for help with photography. One of us (I.N.) would like to thank Prof. F. O. Schmitt and others in his group for encouragement and the use of laboratory facilities in the Department of Biology at M.1.T. during the very early stages of this work. We also thank Dr M. Goldsmith of the Biology Department at the University of Pittsburgh for her constructive suggestions pertaining to an earlier version of this manuscript. This work was supported in part by Grant L-36 from the Health Research and Services Foundation to J.S.
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References BJERS1NG, E. 1967. O n thc u l t r a s t r u c t u r e o f g r a n u l o s a lutein cells in p o r c i n e c o r p u s luteum. Z. Ze!lJbrsch. mikrosk. Anat., 82, 187-21 I. CHRrSTENSEN, A. K. I965. The fine s t r u c t u r e o f testicular interstitial cells in guinea pigs. J. Cell Biol., 26, 911-935. CHRtSrENSEN, A. K. a n d FAWCETT, D. W. 1961. The n o r m a l fine s t r u c t u r e o f o p o s s u m testicular interstitial ceils. J. biophys, biochem. Cytol., 9, 653 670. DROZ, B. a n d KOENtG, H. 1970. L o c a l i z a t i o n o f p r o t e i n m e t a b o l i s m in n e u r o n s . In: Protein Metabolism o / t h e Nervous System (A. L a j t h a , Editor) pp. 93 108. P l e n u m Press, N e w Y o r k . FAWCETT, D. W. 1966. The Cell: An Atlas o f Fine Structure. S a u n d e r s , P h i l a d c l p h i a . FERNAND'6:Z, H . L. a n d DAVlSON, P. F, 1969. A x o p l a s m i c t r a n s p o r t in the crayfish nerve c o r d . Proe. Natl. Acad. Sci. U.S.A., 64, 512-519. KENNEDY, D., SEt,VERSTON, A. 1. a n d REMI ER, M. P. 1969. A n a l y s i s o f restricted n e u r a l n e t w o r k s . Science, N . Y . , 1 6 4 , 1488 1496. LOCKE, M. 1969. T h e u l t r a s t r u c m r e o f the o e n o c y t e s in the m o l t / i n t e r m o l t cycle o f a n insect. Tissue & Cell, 1, 103 154. MALnOTR~_, S. K. a n d MEEK, G. A. 1961. A n E . M . S t u d y o f s o m e c y t o p l a s m i c inclusions o f the n e u r o n e s o f the P r a w n : Leander serratus. Jl R. microsc. Soc., 80, 1 8. MAYNARD, E. A. 1971. E l e c t r o n m i c r o s c o p y o f s t o m a t o g a s t r i c g a n g l i o n iu the lobster Homarus americanus. Tissue & Cell, 3, 137-160. REYNOLDS, E. S. 1963. T h e use o f lead citrate at high p H as an e l e c t r o n - o p a q u e stain in electron m i c r o s c o p y . J. CellBiol., 17, 208 212. Ross, L. S. 1922. C y t o l o g y o f the large nerve ceils o f the crayfish (Cambarus). J. comp~ Neurol., 34, 37 71. S}tANTIhA, T. R., MANOCHA, S. L., BOURNE, G . H. a n d KAPPERS, J. A. 1969. T h e m o r p h o l o g y a n d c y t o l o g y o f n e u r o n s . In: The Structure and Function o / Nervous Tissue (G. H . B o u r n e , Editor), Vol. 2, pp. 1 67. A c a d e m i c Press, N e w Y o r k . SIEKEWTZ, P. 1963. P r o t o p l a s m a : E n d o p l a s m i c r e t i c u l u m a n d m i c r o s o m e s a n d their properties. A. Rev. Physiol., 25, 15 40. TRUJIL;to-CENOZ, 0 . 1962. S o m e aspects o f s t r u c t u r a l o r g a n i z a t i o n o f a r t h r o p o d ganglia. Z. Zellforsch. mikrosk. Anat., 56, 649 682. VAN HARREVELD, A. 1936. A p h y s i o l o g i c a l solution for f r e s h - w a t e r c r u s t a c e a n s . Proc. Soc. exp. Biol., 34, 428 432.
I. NADELHAFT* and J. W. SECHRIST-t
AN U N U S U A L CENTRAL C Y T O P L A S M I C BODY IN LARGE CRAYFISH NEURONS" A LIGHT A N D ELECTRON M I C R O S C O P I C STUDY ABSTRACT. An unusual centrally located cytoplasmic body (~20 micron diameter) sometimes found in large neurons of crayfish abdominal ganglia is described. It is uniquely situated near the terminus of the axoplasmic stream and is surrounded by a lightly staining halo (~ I micron) with some radial striae connecting to the axoplasm. Its composition is primarily membranous, consisting of unbranched tubules and vesicles resembling a type of smooth ER. Scattered islands of polyribosomes, granular Ell, and a few mitochondria are also present. Its location, composition and raetabolic activity (H:Meucine autoradiography) suggest a functional role associated with production of axonal components.
Introduction
THE large n e u r o n s o f crayfish a b d o m i n a l ganglia are characterized by a distinct axoplasmic stream that extends well into the somal cytoplasm. This conspicuous fibrous material sometimes swirls a b o u t the nucleus before dispersing t h r o u g h o u t the cytoplasm (Ross, 1922). In some o f these neurons we have recently observed an unusually large and well defined cytoplasmic structure at the light microscopic level. This structure, a dark staining spherical body ( ~ 2 0 microns in diameter) which may a p p r o a c h the size of the nucleus, occupies a central position in the cell soma. lts position appears to be a focal point of the intracellular axoplasmic stream indicating a possible functional relationship between the two structures. To our knowledge there has been no report in the literature that m e n t i o n s or * VA Hospital (Leech Farm Rd.), Pittsburgh, Pa. 15206, and Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213. t Department of Pharmacology and Anatomy and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213, present address: Department of Anatomy, College of Medicine, University of Arizona, Tucson, Arizona 15213. Manuscript received 24 November 1971.
illustrates a c o m p a r a b l e cytoplasmic structure for either invertebrate or vertebrate neurons. A l t h o u g h the light microscopic cytology of large crayfish neurons has been studied in considerable detail by Ross (1922) and earlier investigators there are very few, if any, published descriptions o f their ultrastructure. Several electron microscopic studies of other crustaceans and a r t h r o p o d s are available (Malhotra and Meek, 1961; Trujillo-Cenoz, 1962; M a y n a r d , 1971), but again there is n o description o f a cytoplasmic b o d y c o m p a r a b l e to that observed in the crayfish. The objective o f this report is to present the results o f light and electron microscopic observations, including preliminary autoradiographic studies, of this p r o m i n e n t cytoplasmic body and its relationship to other cytoplasmic a n d axoplasmic organelles.
M a t e r i a l s and M e t h o d s
Animals
Crayfish (Procambarus clarkii) ranging in size f r o m 3-5 inches were obtained f r o m the L e m b e r g e r Co. in Oshkosh, Wisconsin. They were kept in either cooled tanks (18°C) or at r o o m t e m p e r a t u r e (25"C) for several 235
236 weeks prior to use and were fed a few pellets daily of "Shrimp-el-etts' (Longlife Fish F o o d Products, Harrison, N.J.). The animals selected for use were all in good condition as determined by visual observation. They were anesthetized by exposure to carbon dioxide (about 15 minutes) or by cooling in crushed ice. The abdominal nerve cord was carefully dissected out and processed as described below.
Microscopy Cords were removed, washed in Van Harreveld's ringer (Van Harreveld, 1936) and processed in one of three ways: (a) in Bouin's fluid followed by paraffin embedding, (b) in 5'!; glutaraldehyde in Van Harreveld's Tinger at p H 7 3 for six days with postfixation in I~',; osmium tetroxide in 0.05M phosphate buffer at the same p H for 1 hour followed by dehydration in ascending series of ethanol and embedding in Epon 812, and (c) in 51!; glutaraldehyde in cacodylate buffer (0.067M, p H 7 3 , 6 5 0 milliosmoles) for 3 days followed by postfixation and embedding as in (b) above. Only nerve cords fixed in the latter manner were utilized for electron microscopy. Light microscopic observations were made on both paraffin and epoxy embedded tissue. Several abdominal nerve cords with their six attached ganglia were cut into 8-10 micron paraffin sections and stained with thionin for general observations. The second, third, or fourth ganglion of several additional crayfish cords embedded in Epon were sectioned serially at one micron in the transverse, horizontal, or sagittal planes. These were cut with a diamond knife on a Huxley ultramicrotome and were stained for one minute with 0-5'!,; toluidine blue in 0.5 ~; borax at 60'~C. The epoxy embedded sections, in which the cytoplasmic body was first observed, were used for studying the shape and distribution of the body, for localizing appropriate areas for thin sectioning, and for autoradiographs (see below). Thin sections for electron microscopy were cut on a Porter-Blum MT-1 ultramicrotome with a diamond knife, mounted on F o r m v a r and carbon coated grids, stained for ½ hour with 5 % uranyl acetate, counterstained with lead citrate (Reynolds, 1963) for 2 minutes, and examined in a Phillips--200 electron microscope.
NADELHAFT AND SECHRIST
Autoradiography Crayfish were injected intraganglionically (2rid and 3rd abdominal ganglion; see Fernandez and Davison, 1969) with Ha-L Leucine (5 t~C per ganglion) and were sacrificed 8 days later with the abdominal portion of the nerve cord subsequently processed, embedded in Epon, and cut as described above under Microscopy. Thick Epon sections (one micron) were placed serially on separate water drops on a 1" ", 3" microscope slide and were dried down using a hot plate. These slides were then dipped in liquid enmlsion (Kodak NTB-3 diluted with an equal amount of distilled water) at 45'C, dried, and placed into slide boxes. These were sealed and placed in the refrigerator (6'C) for exposure times of one to three months. Development of exposed slides was done in D-19 developer for 2 min. at 24"C followed by fixing and washing. The dried slides were stained in either toluidine blue or Paragon Multiple Stain (PS 1301), cover slipped, and observed with the light microscope. Observations
Light microscopy A camera lucida drawing (Fig. 1) of a transverse section through the third abdominal crayfish ganglion is included for orientation purposes. Characteristic features of these ganglia are a central neuropil with four giant axons near the dorsal surface and a rind of cell bodies near the ventral surface. In some of the larger cells a prominent spherical cytoplasmic structure can be seen following toluidine blue staining of thick Epon sections of tissue fixed in osmium tetroxide. A typical appearance of this cytoplasmic structure, which we refer to as ~body" for lack of a better term at the present, is shown in Fig. 2. in E p o n sections stained with toluidine blue the body, which is more densely stained than much of the surrounding cytoplasm, is positioned in the center of the neuron and appears to be located near the region where the axoplasmic stream disperses throughout the cytoplasm. In neurons with a diameter of 100 microns it is not unusual for the body to be as large as 20 microns in diameter. It is surrounded by a halo about one micron thick that stains more lightly
CYTOPLASMIC BODY 1N CRAYFISH N E U RO N S than most of the remaining cytoplasm. Light staining strands or striae that are in continuity with a similar staining cytoplasmic network radiate outward from this halo as opposed to the nucleus where the strands can be seen to go around it. This network, in turn, is continuous with the well defined axoplasmic stream, Electron microscopic observations (see subsequent descriptions) reveal that these light staining areas contain fibrillar elements similar to those in the a×oplasm. The appearance of the body from one neuron to another is usually quite similar (Figs. 3-5). Out of approximately 15 examples of such neurons studied in detail thus far in various planes of section only one contains a body which is more oblong than spherical in shape (Fig. 6). In neurons sectioned horizontally, such as the one in Fig. 5, the main axoplasmic stream cannot always be observed because of its position dorsal to the body. However, this plane of section cleaily illustrates the outward radiation of the striae from the body. Other characteristic light microscopic features (Figs. 2 6) of these neurons containing the central cytoplasmic body are numerous crescent-shaped dictyosomes or Golgi bodies, a widely dispersed cytoplasmic substance of intermediate staining density, and a displaced nucleus with one or two prominent nucleoli. Within the intracellular axoplasmic stream thread-like structures which are probably mitochondria can be observed. No Nissl bodies similar to those in large vertebrate neurons are present. However, as will be demonstrated in subsequent electron micrographs, the homogeneous background staining of intermediate density is due to the diffuse distribution of polyribosomes and granular endoplasmic reticulum. The widespread and somewhat isolated distribution of the Golgi bodies in crayfish and other invertebrate neurons also differs from those of vertebrate neurons which often are located in a perinuclear position. It has been suggested that the vertebrate configuration of the Golgi bodies may be partly due to fixation procedures (Shantha, Manocha, Bourne, and Kappers, 1969). The cytoplasmic features of comparable neurons without the body differ little, if at all, from those containing the body. Therefore, there is nothing to indicate D
237
that neurons containing the body are in any way degenerative or pathological. In paraffin embedded sections fixed with either formalin or Bouin's solution instead of osmium tetroxide the surrounding cytoplasm, but not the body, is stained with basic dyes such as thionin or toluidine blue. In at least one instance we located a negatively stained sphere in the central cytoplasmic zone (Figs. 7, 8). Although much of the surrounding cytoplasm is stained, Golgi bodies are not apparent. This indicates that the central body is probably primarily membranous in composition, as are the Golgi bodies, and requires a fixative such as osmium for its preservation.
Topographical distribution Prior to describing the fine structure of the body, a brief account of our current understanding of the geographical distribution of crayfish neurons containing the body is presented. This distribution has been studied by observing serial one micron sections of abdominal ganglia cut in either transverse, sagittal, or horizontal planes. Out of 15 examples studied thus far the body has been found in bilaterally symmetrical neurons in at least two instances. The cells in Figs. 3 and 4 represent one such pair located rostrally in the 3rd ganglion. Such symmetry has not been established as a general property however. For example, after making a series of camera lucida drawings of horizontal sections through the second abdominal ganglion (see Fig. 17), it was determined that out of a total of four neurons containing the body three were located on one side of the midline and only one on the other. A few of the neuron somas containing the cytoplasmic body are located in positions comparable to certain of those which have been mapped for 9 fast flexor motor neurons whose axons exit by way of the third roots (Kennedy, Selverston, and Remler, 1969). In one instance, the axon of such a neuron located in the rostral part of the 3rd ganglion was traced by means of serial sections further rostrally into the nerve cord as it crossed the midline and assumed a dorsolateral position in the nerve cord. Although it was not traced all the way to the third root, this axon probably originated from one of the fast flexor motor neurons innervating the next rostral segment via the third root.
238
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Fig. 1. Camera lucida drawing of a transverse section through the 3rd abdominal crayfish ganglion at the level of emergence of the first pair of roots. Typical features illustrated are a rind of cell bodies adjacent to the ventral ('q) surface and medial Imgl and lateral fig) giant axons near the dorsal (D) surface. The central core or neuropil (Np) contains numerous nerve cell processes of which only a few are shown. A spherical "body' is indicated (B, arrows) in each of two laterally placed nerve cells on the left side of the ganglion. Fig. 2. Light micrograph of a large nerve cell from a region comparable to that indicated by arrows in the above drawing (Fig. I ). In the center of this nerve cell is a prominent darkly stained body (B) which is surrounded by a light halo that is comparable in staining intensity to the axoplasmic stream (As) and a light network distributed throughout the cytoplasm. Other intracellular structures shown at this low magnification are the nucleus (N/ and numerous darkly stained oval dictoyosomes (Golgi bodies). [n the field of view are parts of other nerve cells, glial cell nuclei (*), and a portion of the neuropil (Np). Epon section stained with toluidine blue. × 650. Figs. 3, 4. Light micrographs illustrating two examples of the body as it appears in sagittal sections. Again note the position of the body in relation to the axoplasmic stream and the light staining striae radiating from the body. These two neurons occupied bilateral symmetrical positions in the rostral 3rd abdominal ganglion. Epon sections stained with toluidine blue. × 640. Fig. 5. Light micrograph illustrating an example of the body as it usually appears in horizontal sections. In this neuron (from 2nd abdominal ganglion) and the one in Fig. 6 the main axoplasntic stream is dorsal to the plane of section. The light halo with striae radiating out fi'om it is clearly shown. Note the position of the body with respect to the light staining nucleus. Epon section stained with toluidine blue. >:640. Fig. 6. Light micrograph illustrating an example of an unusually shaped body in a horizontal section. [t is more obloag than spherical. Epon section stained with toluidine blue. :: 400. Figs. 7, 8. Light micrographs of two transverse paraffin sections (7 microns thick) through a large crayfish neuron in the 3rd abdominal ganglion. An intervening section is omitted. In Fig. 7 the body appears as a negatively stained sphere in the center of the basophilic cytoplasm; the unstained region to the left of the body is part of the nucleus. In Fig. 8 the section passes through the middle of the nucleus including a prominent nucleolus: clearly shown is the unstained axoplasmic stream penetrating the cytoplasm with some of it dispersing in the general vicinity of the body shown in Fig. 7. Bonin's fixative, thionin stain. × 740. Fig. 9. Electron micrograph of a strip of cytoplasm between the nucleus (N) below and the body (B) above. The cytoplasm is characterized by a relatively uniform distribution of mitochondria (M), Golgi bodies (G), clusters of polyribosomes (R), and endoplasmic reticulum (ER). The ER is primarily the branched tubular variety with some profiles having a few attached ribosomes while others have none. Ribosome clusters lie between the ER profiles. The body contains membranous vesicles and tubules (see Figs. 10-12). It is surrounded by a halo (H) of agranular cytoplasm. Same cell as Fig. 2. ,. 14,750. Fig. 10. Electron micrograph of a strip of body (B) and adjacent cytoplasm (Cyt) including the intervening transitional region or halo (H). The field of view extends from the middle of the body above toward the cytoplasm below that is between the body and the nucleus. Although the body consists primarily of large vesicles and tubular profiles of smooth endoplasmic reticulum, small islands of granular ER, polyribosomes (R), and an occasional mitochondrion (M) with associated glycogen particles can be observed. The rectangular area is shown at higher magnification in Fig. 12. Same cell as Fig. 2. z 14,750.
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NADELHAFT AND SECHRIST
242 It s h o u l d be e m p h a s i z e d t h a t the ganglia o f s o m e crayfish d o n o t h a v e n e u r o n s c o n t a i n ing t h e b o d y . H o w e v e r , if n e u r o n s w i t h the b o d y are f o u n d in o n e crayfish g a n g l i o n , t h e y are u s u a l l y f o u n d in o t h e r g a n g l i a o f t h a t s a m e crayfish a n d in o t h e r crayfish f r o m the s a m e g r o u p . T h u s far we h a v e s t u d i e d five different g r o u p s o f crayfish. N e u r o n s cont a i n i n g the b o d y were f o u n d in the a b d o m i n a l g a n g l i a o f three o f these five g r o u p s (see Discussion for additional comments).
Electrotl microscopy C y t o p l a s m i c features, o t h e r t h a n the central b o d y , of large crayfish n e u r o n s are generally c o m p a r a b l e to m a n y o t h e r i n v e r t e b r a t e
n e u r o n s at the u l t r a s t r u c t u r a l level (Figs. 9, 13-15). T h e e n d o p l a s m i c r e t i c u l u m is widely d i s t r i b u t e d t h r o u g h o u t the c y t o p l a s m . It c o n s i s t s p r i m a r i l y o f the b r a n c h e d t u b u l a r variety with an irregular attachment of ribosomes. The percentage of ribosomes a t t a c h e d to these m e m b r a n e s is low. C l u s t e r s o f free p o l y r i b o s o m e s d i s t r i b u t e d b e t w e e n the t u b u l a r E R profiles are n u m e r o u s . I n c o n t r a s t w i t h v e r t e b r a t e n e u r o n s the scarcity o f s t a c k e d cisternae o f g r a n u l a r e n d o p l a s m i c r e t i c u l u m is striking. T h i s results in the diffuse b a s o p h i l i a o r b a c k g r o u n d s t a i n i n g o b s e r v e d at the light m i c r o s c o p i c level with the a b s e n c e o f p a r t i c u l a t e N i s s l bodies. O t h e r c y t o p l a s m i c organelles exhibit n o
Fig. I I. Electron micrograph of a region in the interior of the body selected to illustrate an area containing clusters of polyribosomes (R), mitochondria (M), and granular endoplasmic reticulum (gER). Such areas are scattered among a primary population of vesicles (V) and smooth tubular profiles iT). Lightly stained microtubules (rot, arrows) appear to form a loose meshwork throughout the body. Same cell as Fig. 2. ,42,500. Fig. 12. Electron micrograph of a region in the interior of the body (see rectangle in Fig. 10) with the typical distribution of vesicles (V) and smooth tubular profiles IT). The diameter of those structures appearing as circles in cross section ranges between 300 and 2500~ whereas the tubules range between 500 and 1000 .&, The large circles (probably vesicles) generally have an empty appearance except for sometimes containing one or several smaller vesicles. Occasionally the tubules have bulbous ends (large arrow). Microtubules (nat) about 230 ~. in diameter are present in the background; they also may appear to be continuous with the larger tubular structures (double small arrows/. In the upper right corner some typical branched, smooth endoplasmic reticulum (sER) can be seen. Same cell as Fig. 2. , 42,5(10. Fig. 13. Electron micrograph of a large crayfish neuron whose cytoplasmic body (B) and surrounding halo (HI is in direct continuity with part of the main intracellular axoplasmic stream (As, large bidirectional arrows) as well as smaller branches (small arrows) of it. Note vesicles (V) similar to those in the body within the axoplasmic stream. Adjacent to the axoplasm and the halo surrounding the body are typical cytoplasmic components such as endoplasmic reticulum (ER), polyribosomes, and mitochondria (M). The small dense particle~ in the axoplasm are probably glycogen. , 14,750 Fig. 14. Electron micrograph of a crayfish neuron in which much of the axoplasmic stream (As, large bidirectional arrows) by-passes the body (B) as it disperses throughout the cytoplasm. Large fingers of axoplasm (small arrows), however, are in continuity with the halo (H) surrounding the body. Fibrillar elements shown to be microtubules (rot) at a higher magnification can be seen in the axoplasm and its finger-like branches. Several Golgi bodies (G) as well as endoplasmic reticulum (ER), (polyribosomes (R), and mitochondria (M) are present in the adjacent cytoplasm. / 14,750. Fig. 15. Electron micrograph of transition region between the axoplasmic stream and cytoplasm of a large crayfish neuron without a body. The distribution of cytoplasmic organelles such as endoplasmic reticulum (ER), polyribosomes (R), mitochondria (m), and Golgi bodies (G) with associated vesicles are comparable to neurons with the body. Note the numerous microtubules in the intracellular axoplasmic stream (As, bidirectional arrows). × 24,000. Fig. 16. Electron micrograph of a portion of the intracellular axoplasmic stream (As) as it skirts the nucleus (N). The axoplasm contains vesicles, endoplasmic reticulum (ERL and mitochondria (M) in addition to numerous microtubules. Same cell as Fig, 15 above. × 24,000
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246 particularly unusual features. The Golgi bodies appear in fixed tissue as large crescents of flattened sacs or cisternae with numerous associated vesicles (Figs. 9, 14 and 15). They are extensively distributed in a somewhat isolated fashion throughout the cytoplasm with the exception of the central cytoplasmic body and the intracellular axoplasmic stream. The close orientation of some of the Golgi bodies to the axoplasmic stream (Fig. 14) is such that vesicles can easily be ieleased into it. It may be significant that the Golgi bodies vary in size and that some neurons without a central body contain larger Golgi bodies. The mitochondria are numerous with relatively few cristae which sometimes appear to be disrupted by swelling during the fixation process. Dense particles, probably glycogen, are occasionally associated with them (Fig. 10) Sometimes the glycogen particles can be observed adjacent to membrane bound structures filled with electron dense granular material (Figs. 13, 14). These may represent transformed mitochondria since they are similar in size. Typical microtubules are dispersed among the cytoplasmic structures described above and are especially evident in regions described as a light cytoplasmic network at the light microscope level. The large cytoplasmic body, at low magnification (Fig. 10), appears to consist primarily of membranous elements preserved as round vesicles and unbranched tubular structures which are most likely a type of smooth endoplasmic reticulum. These membranous elements are embedded in a lightly stained cytoplasmic matrix that is continuous with the halo which separates the body from the surrounding cytoplasmic organelles. Within the body a few mitochondria and scattered islands of polyribosomes and granular ER can be observed. The latter structures as well as a loose background meshwork of microtubules are better shown at higher magnification (Fig. 11 ). Closer examination of the membranous elements within the body (Figs. 11, 12) reveals that the tubular structures range in diameter from 500 to 1000 ~ . They exhibit no particular orientation within the body. Without extensive serial sectioning it is difficult to determine the length of these tubules or the extent to which they are continuous or separate interwoven cylindrical structures. The majority, however, do not
N A D E L H A F T AND SECHRIST appear to be the typical branched, interconnected tubules most commonly referred to as smooth endoplasmic reticulum. The content of many of these membrane bound tubules is more electron-dense than the surrounding cytoplasm. Occasionally 230 microtubules appear to be continuous with the larger tubular profiles (Fig. 12), but this may be due to overlap within the section rather than actual continuity. Those membrane bound elements within the body which appear to be circular in section (Figs. 1 I, 12) have diameters in the range of 300 to 2500 ~. Scrutiny of the density of material within these circular profiles reveals that most of the smaller diameter circles have a core which is more electron-dense than the background cytoplasm whereas the content of the larger circles is comparable in density to the background cytoplasm with the exception of an electron dense rim adjacent to the surrounding membrane. It would appear that many of the smaller electron-dense circles represent transversely cut tubules since their diameters and electron density are equivalent. The larger circles, on the other hand, are most likely spheres or vesicles. A few of them contain one or more small vesicles. The outer electron-dense rim within the vesicular membrane may be partly due to the inclusion of a portion of the spherical shell within the section ( ~ 1000 ~ thick). A short calculation indicates that a 1000 A section through the middle of a 2000 A sphere would have its edge broadened to a width of approximately 150 ~. An alternative explanation for the presence of an electrondense rim in the larger vesicles is that these spheres may result from "ballooning" of tubules whose electron dense contents cleave in the middle but adhere to the outer membrane. Sometimes the tubules do have bulbous ends (Fig. 12) which would appear to be isolated spheres or vesicles if cut in the perpendicular plane. The distinct intracellular axoplasmic stream that penetrates deeply into the cell soma is composed of numerous microtubules, mitochondria, smooth ER, and vesicles of various sizes (Figs. 15 and 16). Of particular interest in those cells that contain the body is the apparent relationship of the axoplasmic stream to the body. Either part of the main stream may directly
CYTOPLASMIC BODY IN CRAYFISH N EU RO N S approach the body (Fig. 13) or branches of the stream may be given off to the body (Fig. 14) as it disperses throughout the remainder of the cytoplasm. Some of the vesicles in the axoplasmic stream are comparable to those in the central body as well as those associated with the Golgi bodies. It is clear that the light halo surrounding the body and similar light staining areas observed in the cytoplasm at the light microscope level contain microtubules that are in continuity with those in the axoplasmic stream. Neurofilaments such as those seen in vertebrate axons have not been observed although a detailed study has not been made at high magnification.
A utoradiography The observations reported here are taken from material belonging to an unrelated experiment and are therefore of a fragmentary nature. At the present time available autoradiographs of crayfish neurons containing the central body are limited to an 8 day time interval following intraganglionie injection of Ha-leucine. Since these provide useful preliminary information on the metabolic activity of the body, it was deemed appropriate to present them. The autoradiographs shown in Figs. 17-20 were taken after 30 days exposure time. Alternate sections exposed for 90 days revealed such a dense distribution of silver grains that their usefulness for grain counting over neurons was limited. In general, nearly all nerve cells, including those containing the body, revealed high levels of incorporation of H'Meucine. The density of silver grains over the nuclei was slightly lower than over the cytoplasm while nucleoli were significantly more heavily labelled. Axons in the neuropil and nerve cord which originated from cells in the injected ganglion were labelled, but not those from other ganglia. However, the Schwann cell sheaths of all axons within the vicinity of the injected ganglion were labelled irrespective of the origin of the axons. At first glance the grain density over the cytoplasmic body appeared to be comparable to that of the remaining cytoplasm, but more detailed studies of grain counts revealed the density over the body to be slightly lower than the average over the cytoplasm. For example, counts made on four serial sections through the body gave an average of 204- 7
247
counts over the body and 231 ~5 counts within equivalent areas (3 from each section) of the surrounding cytoplasm. For the above measurements an 18.9 micron diameter circle with an area of 282 square microns was used. Closer examination of certain sections through the body at higher magnification reveals some central spots with relatively high grain density (Fig. 18). These could correspond to those areas that contain islands of polyribosomes and granular ER shown in Figs. 10 and 11. However such regions of high and low grain density within the body can also be due to statistical fluctuations in the number of radioactive nuclei which have decayed. The latter possibility is supported by examination of alternate sections (Figs. 19, 20) which reveal considerable variability in grain distribution. Furthermore, examination of sections exposed for 90 days with much higher grain density reveals less of a differential from one area to another. It would appear, therefore, that eight days after injection of H:Meucine the central cytoplasmic body indicates incorporation of labelled precursor at levels nearly equivalent to the surrounding cytoplasm. This clearly establishes the fact that the body is quite active metabolically. Discussion
Agranular or smooth endoplasmic reticulum has been known for some time to have a widespread occurrence in cell types of very different function (Christensen and Fawcett, 1961). To our knowledge, however, there are no published descriptions of invertebrate or vertebrate neurons that contain an aggregate of smooth ER and vesicles comparable to that which we have described in the large cytoplasmic body of certain crayfish neurons. The axons of most nerve cells contain some smooth ER, but within the cell some various configurations of granular ER as well as Golgi bodies are normally the predominant membranous structures. Since smooth ER is not abundantly found in neurons, descriptions dealing specifically with its distribution and potential functional significance in various other cell types are of particular interest. A comparison of representative electron micrographs of
248
NADELHAFT AND SECHRIST
s m o o t h E R a n d the different functional roles a t t r i b u t e d to it in various cells clearly reveals t h a t generalizations c a n n o t be made f r o m one cell type to another. A few of the proposed roles for s m o o t h E R include detoxification m e c h a n i s m s as well as lipid a n d cholesterol m e t a b o l i s m in liver, lipid t r a n s p o r t in intestinal epithelium, biosynthesis of steroid h o r m o n e s in cells of the testis, ovary, a n d adrenal glands, a n d excitation-contraction coupling in skeletal muscle (Fawcett, 1966). A recent electron microscopic study of oenocytes of an insect led to the suggestion t h a t those cells c o n t a i n i n g densely packed s m o o t h E R could be the source of ecdysone a n d the stimulus for m o l t i n g (Locke, 1969). W h a t e v e r the putative functional role these m e m b r a n o u s elements have in various cells, it has been well d o c u m e n t e d that their degree of d e v e l o p m e n t a n d the presence of associated enzymes m a y change m a r k e d l y d u r i n g physiological or e n v i r o n m e n t a l fluctuations (Siekevitz, 1963; Bjersing, 1967). A l t h o u g h we have n o t been able to d e t e r m i n e the functional significance of the large cytoplasmic body in crayfish neurons, certain features of this p r e d o m i n a n t l y m e m b r a n o u s structure have led us to a hypothesis concerning its role. The position o f the b o d y in the cell near the t e r m i n u s of the axoplasmic stream and its c o m p o s i t i o n suggest a function associated with p r o d u c t i o n of certain axonal constituents. The body is s u r r o u n d e d by a halo of light staining material with similar staining striae r a d i a t i n g outward. Some of these light staining areas clearly merge with the main axoplasmic stream whereas others diffuse t h r o u g h o u t the cell soma. Closer e x a m i n a t i o n
at the ultrastructural level reveals such areas contain microtubules which are apparently c o n t i n u o u s with those in either the m a i n axoplasmic stream or those dispersed t h r o u g h o u t the cytoplasm. Such light staining areas m a y represent pathways whereby the body, cytoplasm, a n d axoplasmic stream are interconnected. The body is c o m p o s e d primarily of m e m b r a n o u s elements consisting of spherical vesicles a n d tubules which resemble a type of u n b r a n c h e d , s m o o t h endoplasmic reticulum in b o t h form a n d dimensions. W i t h i n the central p o r t i o n of the b o d y there are also some, but n o t extensive, a m o u n t s of ribosomal elements. These ribosomes are usually f o u n d in scattered small islands c o n t a i n i n g g r a n u l a r ER, clusters of polyribosomes, and a few m i t o c h o n d r i a . T h e m e m b r a n e s of the granular E R a p p e a r to be c o n t i n u o u s w i t h the s u r r o u n d i n g s m o o t h ER. Microtubules f o r m a loose m e s h w o r k t h r o u g h o u t the body in a d d i t i o n to being present in the halo a r o u n d the body. Some of the vesicles a n d s m o o t h E R observed in the axoplasm are c o m p a r a b l e to those of the outer p o r t i o n of the body. Morphologically it would appear, especially in several instances where large fingers of a x o p l a s m a p p r o a c h the body, t h a t some m e m b r a n o u s elements of the b o d y such as vesicles move into the axoplasmic stream from the body where they are formed. The morphological relationships of various structures within the body are not clear. Occasionally a few of the tubular structures have swollen ends which may represent vesicle formation. F r o m other studies, however, it is k n o w n that tubules of s m o o t h e n d o p l a s m i c reticulum are fragile a n d sensitive to fixation procedures. For example,
Fig. 17. Autoradiograph of a horizontal section through the 2nd abdominal ganglion of a crayfish sacrificed 8 days after intraganglionic injection of 5 ~C of HZ~-leucine. The rostral (R) and caudal (C) ends of the ganglion are indicated. The cell identified by the arrow contains a body which is shown at higher magnification in Fig. 18. Closer examination of the distribution of silver grains at intermediate magnification reveals the cytoplasm to be heavily labelled while the intracellular axoplasm, the body, and nucleoplasm, except for the nucleolus, are labelled at somewhat lower levels. Serial sections of this ganglion indicated that four neurons contained the body: a rostral one (arrow) on the right side and three others (not shown) on the left side including a caudal-lateral one photographed in Fig. 5. Emulsion covered Epon section, 30 day exposure, Paragon Multiple Stain. ~: 115.
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Fig. 18. A u t o r a d i o g r a p h at h i g h e r m a g n i f i c a t i o n o f the n e u r o n with a c y t o p l a s m i c b o d y indicated by an a r r o w in Fig. 17. G r a i n c o u n t s over the d a r k l y stained b o d y are slightly less t h a n in e q u i v a l e n t sized regions in the r e m a i n i n g c y t o p l a s m (see O b s e r v a t i o n s for details). T h e overall d i s t r i b u t i o n o f grains a b o v e b o t h the c y t o p l a s m a n d the b o d y is quite u n i f o r m a l t h o u g h a r e a s with g r e a t e r g r a i n density c a n be observed. ~ 625. Figs. 19, 20. A u t o r a d i o g r a p h s o f serial sections o n either side o f t h a t s h o w n in Fig. 18 to illustrate the variability o f g r a i n d i s t r i b u t i o n over the b o d y f r o m o n e section to a n o t h e r . In Fig. 18 m a n y o f the grains are c o n c e n t r a t e d centrally in a r e a s k n o w n to c o n t a i n r i b o s o m e s a n d g r a n u l a r E R b u t in Figs. 19 a n d 20 n u m e r o u s g r a i n s are also over p e r i p h e r a l p a r t s o f the b o d y . :~:625.
250 in interstitial cells of the guinea pig testis, the tubules of ER are sometimes preserved as vesicles of various sizes if glutaraldehyde is not used as a fixative (Christensen, 1965). Because glutaraldehyde was used routinely in our procedures, the vesicles in the cytoplasmic body of crayfish neurons probably more closely represent the true configuration rather than fixation artifacts although the latter possibility cannot be ruled out. In several instances 230 ~ microtubules have also been demonstrated as appearing continuous with the larger tubular profiles of smooth ER within the body. The latter observation may be significant with respect to the origin of microtubules or transport of materials but we have no evidence for either possibility. The results from autoradiographic studies clearly establish that the central cytoplasmic body is metabolically active. Eight days after injection of H'~-leucine a considerable number of silver glains aie more or less evenly distributed over the entire body. In fact, the level of activity (per unit area) is approximately equal to that of the remainder of the cytoplasm. The question of whether the body is a primary site of synthesis or plays an intermediate role in which it is the recipient of proteins produced elsewhere remains unanswered. The body could be a special packaging station, similar to that of the Golgi apparatus (Droz and Koenig, 1970), where proteins produced in the cytoplasm are modified prior to being transferred to the axoplasmic stream. However, the presence of areas with ribosomes within the body suggests that at least some protein synthesis must occur there as well. Nevertheless the relative paucity of ribosomes compared with the abundance of smooth ER would appear to support the hypothesis of the body serving primarily as an intermediate station for modification or assembly of molecules (structural or secretory) destined for the axon. Further examination of such questions dealing with the initial site of incorporation of labelled precursors, the nature of the final products, and the extent of movement of molecules between the body, cytoplasm, and axoplasm requires additional autoradicgraphic experiments utilizing shorter time intervals between injection of labelled precursor and sacrifice. However, as pointed out below, we aie hampered by the incon-
N A D E L H A F T AND SECHRIST sistent frequency of the body"s occurrence. This difficulty should be resolved before proceeding with additional incorporation studies. The study of functional and metabolic properties of this body under predictable and controlled conditions has been made difficult by our inability to be certain of its occurrence within any particular neuron or any individual crayfish. We have found examples of the body in three out of five different groups of crayfish. Some of the groups with the body were obtained as much as one year apart in experiments carried out in different laboratories. In attempts to differentiate between those groups in which bodies were found and those where they were not found, we have noted the following situations. In two cases where bodies were found the temperature of the water in which the animals were kept was different ( 2 5 C in one case and 18:'C in the other). The abdominal nerve cords were collected following two different kinds of anesthesia, CO2 and cold, and again bodies were found in animals treated by each method. In one group animals in which no bodies were found, the fixation procedure was different from the others in that a lapse of approximately 3½ months occurred between glutaraldehyde fixation and osmium postfixation. These cords showed much less blackening of membranes than did those cords in which osmium followed glutaraldehyde by only a short time. In this group our inability to locate neurons with the body may be due to the fact that membranous structures including Golgi bodies simply could not be clearly recognized. Although we are unable, at present, to predict the circumstances under which the abdominal ganglion neurons will contain these bodies, we have not yet made any extensive investigation of the many possible variables which could be important. Perhaps the presence of the body is related to a cyclic process such as molting with increased metabolic requirements due to growth. Another possibility is a change in the ion composition of the water in which the animals are stored or the length of time during which they are held before use. Also, the type of food used or the amount provided may be significant. Experiments to investigate these variables are being planned prior to further examination of the metabolic
251
CYTOPLASMIC BODY IN CRAYFISH N E U R O N S activity and function of this unusual cytoplasmic body found in some crayfish neurons.
Summary At the light microscope level large neurons of crayfish abdominal ganglia are characterized by dispersed Nissl substance, numerous prominent Golgi bodies, and a distinct intracellular axoplasmic stream. Observations of tissue postfixed in osmium reveal some of these neurons contain a a darkly stained central cytoplasmic body ( ~ 2 0 microns in diameter) that may approach the size of the nucleus. This well defined spherical body is surrounded by a light staining halo with outward radiating striae some of which are in direct continuity with the axoplasmic stream. Electron microscopic studies indicate the body is primarily membranous consisting of a somewhat isolated aggregate of unbranched tubular structures (500 1000~ in diameter) and spherical structures or vesicles (300-2500 ~ in diametert which together resemble a type of smooth ER. The content of the tubular structures is more electron dense than the background cytoplasm. Within the central region of the body among the tubules and vesicles are scattered islands of polyribosomes, a few mitochondria, and some granular ER. Scattered microtubules (230 ~ ) form a loose background meshwork some of these microtubules appear continuous with
those in the surrounding halo as well as those in the striae that approach the main axoplasmic stream. Many of the vesicles and smooth ER in the axoplasm are similar to those on the peripheral margin of the body. Preliminary HMeucine autoradiography reveals incorporation levels within the body to be nearly equivalent to that of the surrounding cytoplasm. The position of the body near the terminus of the axoplasmic stream, its composition, and its inconsistent presence suggest a cyclic functional role associated with production of certain axonal constituents during undetermined physiological or environmental fluctuations.
Acknowledgements We thank Linda Rose and Arnitia Allison for histological assistance and Charles Abbott and Donald Ferrera for help with photography. One of us (I.N.) would like to thank Prof. F. O. Schmitt and others in his group for encouragement and the use of laboratory facilities in the Department of Biology at M.1.T. during the very early stages of this work. We also thank Dr M. Goldsmith of the Biology Department at the University of Pittsburgh for her constructive suggestions pertaining to an earlier version of this manuscript. This work was supported in part by Grant L-36 from the Health Research and Services Foundation to J.S.
252
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