The tridimensional structure of Nissl bodiesA stereoscopic study in ventral horn cells of rat spinal cord.код для вставкиСкачать
THE ANATOMICAL RECORD 207539-546 (1983) The Tridimensional Structure of Nissl Bodies: A Stereoscopic Study in Ventral Horn Cells of Rat Spinal Cord ALAIN BEAUDET AND ALAIN RAMBOURG Montreal Neurological Institute, McGill Uniuersity, Montreal, Quebec, Canada H3A 2B4 (A.B.) and Dkpartement de Biologie du CEA, C.E.N., Saclay, Gifsur-Yvette, 91-191 cedex, France (A. R.) ABSTRACT The tridimensional structure of rough endoplasmic reticulum was examined with both high and low voltage electron microscopes in large ventral horn cells of rat spinal cord, by combining stereoscopic techniques with the use of thick sections selectively impregnated with heavy metal salts. In all neurons examined Nissl bodies appeared as well defined clusters of densely stained and profusely anastomosed plate-, ribbon-, and thread-like cisternae. Plate-like cisternae were variable in size, often showed a shallow curvature, and usually ran in short parallel arrays, separated from one another by fairly constant intervals. All gave rise at their edges to several ribbon-like extensions which occasionally decreased in width distally, turning into thin, thread-like cisternae. Characteristically, these ribbon-like structures would emerge at a n angle from their plate of origin and smoothly curve away from the plane of the plate to merge with ribbons or threads arising from adjacent or more distant plates. Most plate-like cisternae were found a t the periphery of Nissl bodies and tended to be oriented parallel to their surface. In contrast, the center of NissI bodies was almost exclusively occupied by a complex network of ribbonand thread-like cisternae. It is suggested that the basic platelribbon association here described in spinal motoneurons might be a constant feature of Nissl body architecture in various neuronal types, while the size, orientation, and relative proportion of plate-like cisternae may vary according to the metabolic state and/or functional specialization of the cells. In 1894, Nissl reported the presence of dense basophilic masses within the perikarya of nerve cells. These Nissl bodies have long since been shown to correspond in the electron microscope to compact aggregates of rough endoplasmic reticulum (RER) cisternae (Palay and Palade, 1955). Information is still lacking, however, on the precise shape and spatial arrangement of these RER cisternae, which until recently could only be inferred from the reconstruction of serial thin sections. Cytochemical techniques have now been developed (Thiery and Rambourg, 1976; Tsukita and Ishikawa, 1976; Thiery, 1979) which provide selective staining of intra-cellular membrane systems, to the exclusion of other cellular elements such as cytoskeleton or ribosomes. Such methods make it possible to examine the endoplasmic reticulum in relatively thick sections and, through the use of stereoscopic techniques, to directly visualize its tridimensional architecture. Cci 1983 ALAN R. LISS, INC This approach has already contributed to our understanding of the topographic organization of smooth endoplasmic reticulum in axons of rat spinal and chick ciliary ganglia (Droz et al., 1975; Markov et al., 1976, Rambourg and Droz, 19801, rat neurohypophysis (Alonso and Assenmacher, 1978, 1979a) and mice sciatic and phrenic nerves (Tsukita and Ishikawa, 19761, and of RER in nerve cell bodies of rat spinal ganglia (Droz et al., 1975; Thiery and Rambourg, 1976; Rambourg et al., 19831, supraoptic nucleus (Alonso and Assenmacher, 1979131, nucleus raphe dorsalis (Gamrani and Galas, 1980; Araneda et al., 19801, and substantia gelatinosa (Hannah, 1978)., None of these studies, however, has yielded a comprehensive description of the tridimensional structure of Nissl bodies. The present report is a n attempt a t providing Received February 21, 1983; accepted July 25, 1983 540 A. BEAUDET AND A. RAMBOURG such a description, using as a model the large ventral horn cell of rat spinal cord. MATERIALS AND METHODS The central nervous system of adult male Sprague-Dawley rats was fixed by intra-aortic arch perfusion of a 3.5% glutaraldehyde solution in 0.1 M sodium cacodylate buffer. The spinal cord was dissected out of the vertebral canal and sliced a t the level of the cervical enlargement into 1-mm-thick slabs which were post-fixed for 1 hr in the same fixative. Small tissue blocks (1mm3) including the ventro-lateral aspect of the anterior horn were cut out under a dissecting microscope, rinsed in cacodylate buffer, and stained for 1 hr in a 5% aqueous uranyl acetate solution, at 42°C. Following several rinses in distilled HzO, the specimens were poststained in a lead citrate, copper sulfate, and trisodium citrate solution for 1 h r a t 42°C (Ur-Pb-Cu stain, Thiery and Rambourg, 1976).The blocks were then washed again in distilled H20, post-fixed overnight in a 1% aqueous solution of osmium tetroxide a t 4"C, dehydrated in graded ethanols, and embedded in Epon. Thick (2-5 pm) and semi-thin (0.5-1 pm) sections were cut from each block on a Porter Blum ultramicrotome, deposited on Formvar-coated copper grids, and respectively examined a t high (1000 kV) and low (100 kV) voltage in the electron microscope. Large multipolar neurons were systematically photographed a t magnifications ranging from 2800 to 22,000. Most of these cells undoubtedly corresponded to motoneurons, but the possibility that our sampling also comprised a number of large propriospinal neurons (Cajal, 1909-1911; Aitken and Bridger, 1961)cannot be formally excluded. For stereoscopy, two photographs of the same field were taken a t plus and minus 7" angles from the original 0" position, by tilting the goniometric stage of the electron microscope. Tridimensional visualization was obtained by looking at properly adjusted pairs of such pictures with a stereoscopic binocular lens. RESULTS At low magnification using either conventional or high voltage electron microscopes, the perikaryon shows well individualized aggregates of heavily stained and profusely anastomosed lamellar elements (Fig. 1).These aggregates, which correspond to the blocklike basophilic structures identified as Nissl bodies in the light microscope, present roughly circular or oval outlines in 0.5 pmthick sections but are found to be ovoid in shape when stereoscopically examined with the high voltage electron microscope in 3-5 pm-thick sections. Their internal lamellar constituents were shown in a previous publication (Rambourg et al., 1983) to correspond to rough ER cisternae filled up with heavy metal precipitate. The Nissl bodies are separated from one another by strands of cytoplasm containing densely stained mitochondria, stacks of Golgi saccules and scattered lysosomal granules. In between these organelles, smooth ER cisternae form a pervasive network of delicate, filamentous elements which are continuous with the rough ER cisternae of the Nissl bodies (Figs. 1-3). Although generally well demarcated by these strands of cytoplasm, Nissl bodies may occasionally be confluent and occupy large, irregular regions of the perikaryon (Figs. 1-21. Within each Nissl body, the Ur-Pb-Cu impregnated RER cisternae appear as either plate-, ribbon-, or thread-like structures (Figs. 3-61, The plate-like cisternae are variable in size and shape; they usually show a shallow curvature and may be partially twisted (Fig. 5). These plates are not fenestrated, but give rise a t their edges to several ribbon-like extensions which usually decrease in width distally, becoming narrow, thread-like connecting cisternae. Such ribbon-like structures emerge a t a n angle from their plate of origin, curving smoothly above or below the plane of the plate to merge with adjacent plate-like or ribbon-like cisternae (Figs. 3-5). In each Nissl body, plate-, ribbon-, or thread-like cisternae assume a characteristic tridimensional arrangement, illustrated by the drawing in Figure 4. The plate-like cisternae usually run in short parallel arrays of two or three elements, separated from one another by fairly constant intervals of approximately 200 nm (Figs. 3-5). These stacks of cisternae are frequently, but not exclusively, oriented parallel to the surface of the Nissl body, with the concavity of the plates facing inward. Such a concentric organization can be best appreciated in sections passing through the center of the Nissl body (Figs. 2-4). Each of these plate-like cisternae is connected to several neighbouring plates from either the same or adjacent stacks by means of their lateral ribbon- or thread-like exten- 3-D STRUCTURE OF NISSL BODIES Fig. 1. The perikaryon of a large motoneuron from the anterior horn of rat spinal cord. 0.5 pm-thick, UrPbi Cu-stained section-100 kv, x 10,000. The Nissl bodies (Nb)appear as well delineated clusters of densely stained and profusely anastomosed elements. Between the Nissl 541 bodies, pale strands of cytoplasm exhibit mitochondria (MI, Golgi stacks (G),lysosomes (L), and a loose network of tenuous, thread-like structures which corresponds to the smooth endoplasmic reticulum (arrows). 542 A. BEAUDET AND A. RAMBOURG 543 3-D STRUCTURE OF NISSL BODIES sions (Figs. 3-5). As described above, these ribbon- or thread-shaped cisternae characteristically leave the plane of the plate from which they arise to either directly reach the plate located above or below, or to merge with ribbons or threads originating from more distant plates of the same or adjacent stacks (Figs. 3-6). The consequence of this arrangement is that in the space between two adjacent stacks, the ribbon- or threadshaped anastomotic cisternae show a zig-zag or criss-cross pattern (Figs. 3-51. In some instances, and particularly towards the center of the Nissl body, narrow ER cisternae may accumulate, branching and interconnecting in all directions to form a n elaborate tridimensional network (Fig. 6). The cisternae never seem to interlace or interlock like the links of a chain, but instead appear to be arranged in such a way as to be separated from one another by a fairly constant distance equivalent to that separating the plate-like cisternae within the stacks ke., approximately 200 nm). Figs. 2-6. These figures are stereopairs. Each pair is composed of photographs of the same field taken at two different angles (+7” from the original 0” position of the goniometric stage). A single stereoscopic image may be obtained by using a stereoscopic binocular lens, the distance between the central axes of the two lenses being adjusted to 65-70 mm. All figures are taken from 0.5 jim-thick sections of the ventral horn of rat spinal cord impregnated with heavy metals (Ur-Pb-Cu). Fig. 2. Section from the perikaryon of a large motoneuron showing a collection of interconnected Nissl bodies. Some are directly confluent (e.g., Nb 1and 2);others are separated by a strand of cytoplasm (e.g., Nb 1and 31, yet connected by means of thin, thread-like cisternae of smooth endoplasmic reticulum (arrows). All Nissl bodies are made up of short plate-, ribbon-, and thread-like cisternae anastomosed in all directions. Note that most plate-like cisternae tend to be perpendicular to the plane of section in Nissl bodies cross sectioned in their center (Nb 1,3), but parallel to that plane in Nissl bodies intersected at their periphery (Nb 2). x 6,500. Fig. 3. The overall spatial arrangement of RER cisternae may be appreciated in this tridimensional overview of a single, ovoid Nissl body. Plate-like cisternae are organized into a number of short parallel arrays (large arrows). Characteristically, plates of the same or adjacent stacks are interconnected through flat and narrow, ribbon-like cisternae (small arrows). These ribbonlike extensions occasionally decrease in width, giving rise to thin, thread-like cisternae (arrowheads). Most plate-like cisternae are found at the periphery of the Nissl body, the center being almost exclusively occupied by a complex network of anastomosed ribbon- and threadlike cisternae. x 12,500. DISCUSSION The present study provides a new outlook on the shape and tridimensional organization of RER cisternae within Nissl bodies. In contrast to previous descriptions, which were based on the analysis of serial thin sections (Palay and Palade, 1955; for review, see Peters et al., 1976), the model proposed here relies on direct stereoscopic examination of RER in thick sections, using either high or low voltage electron microscopes. In all neurons examined, Nissl bodies were found to be made up of lamellar or “platelike” cisternae and narrower “ribbon-like” and “thread-like” cisternae. These observations broadly conform with those of Palay and Palade (1955) who originally described “tubules” and “large flattened vesicles” designated as cisternae, as basic components of the Nissl bodies membrane system. As correctly forseen by these and other investigators (for review, see chapter V in Peters et al., 19761, all of these elements are interconnected in a continous ti*idimensional network. Our findings, however, reveal a spatial organization which differs in several respects from that proposed by Palay and Palade (1955). This can be best appreciated by comparing their model of the tridimensional arrangement of RER in Nissl bodies of motoneurons (text Fig. 1 in Palay and Palade, 1955) with our own drawing in Figure 4. In both diagrams, flattened or plate-like cisternae are of different sizes and shapes and form regularly spaced parallel arrays. However, in our diagram, plate-like cisternae are neither fenestrated nor involved in the formation of “reticular sheets,’’ i.e., interconnected with other plates running in the same plane. In fact, ribbon- and threadlike connecting cisternae are never found to remain in or leave at a perpendicular angle, the plane of their plate of origin. A recent analysis of the fine structure of six types of neurons in rat dorsal root ganglia (Rambourg et al., 1983) suggests that the basic platelribbon association here described in spinal motoneurons may be a constant feature of Nissl body architecture. The combination of “tubular” and “lamellar” elements visualized with the same technique in neurons of rat supraoptic nucleus (Alonso and Assenmacher, 1979b) is also in keeping with this interpretation. In contrast, the overall spatial arrangement of RER cisternae appears to vary, not only from one cell type to 544 A. BEAUDET AND A. RAMBOURG Fig. 4. Ink drawing of a Nissl body as seen with a stereoscope in the underlying stereo pair. Note the roughly concentric orientation of plate-like cisternae at the periphery of the Nissl body and, within its core, the complex tridimensional organization of ribbon- and thread-like elements. x 17,000. 3-D STRUCTURE OF NISSL BODIES Fig. 5. The structural relationships between plateand ribbon-like cisternae seen at high magnification. Three parallel plate-like cisternae, roughly perpendicular to the plane of section, occupy the center of the field. Each gives rise to a number of ribbon-like extensions, which gently curve away from the plane of the plate and establish connections with neighbouring plate- (arrows) or ribbon-like (arrowheads)cisternae. ~ 5 0 , 0 0 0 . 545 Fig. 6. The center of a Nissl body seen at high magnification. It is almost exclusively composed of ribbon(arrows) and thead-like (arrowheads) cisternae, anastomosed in all directions. Note that all cisternae remain separated by a fairly constant interval (approx. 200 nm), similar to that separating plate-like cisternae within a stack. X22,OOO. 546 A. BEAUDET AND A. RAMBOURG another, but also within a given category of neurons. Thus the differences in the size, orientation, and relative proportion of plate-like cisternae observed betwen lower motor and various types of primary sensory neurons (Rambourg et al., 1983) or the shift from “lamellar” to “tubular” configurations reported to occur upon dehydration in cells of rat supraoptic nucleus (Alonso and Assenmacher, 1979b). Such variations presumably reflect differences in the functional specialization and/or metabolic activity of the cells. ACKNOWLEDGMENTS This investigation was supported by the Commissariat a l’energie atomique (France), the Medical Research Council (Canada), and a France-Quebec exchange program. The authors are indebted to Dr. Yves Clermont for his inspiring review of the manuscript. They also thank Dr. Alain Marraud and his colleagues for their help with the work a t the high voltage electron microscope (CNRSONERA, Ch5tillon-sous-Bagneux, France), Miss Eva Schneider for preparing the drawing in Figure 4, and Miss Beverley Lindsay for typing the manuscript. LITERATURE CITED Aitken, J.T., and J.E. Bridger (1961) Neuron size and neuron population density in the lumbosacral region of the cat spinal cord. J. Anat., 9538-53. Alonso, G., and I. Assenmacher (1978) The smooth endoplasmic reticulum in neurosecretory axons of the rat neurohypophysis. Biol. Cellulaire, 32:203-206. Alonso, G., and I. Assenmacher (1979a) The smooth endoplasmic reticulum in neurohypophysial axons of the rat: Possible involvement in transport, storage, and release of neurosecretory material. Cell Tissue Res., 199t415-429. Alonso, G., and I. Assenmacher (1979b) Three-dimensional organization of the endoplasmic reticulum in supraoptic neurons of the rat. A structural functional correlation. Brain Res., 170:247-258. Araneda, S., H. Gamrani, C. Font, A. Calas, J.F. Pujol, and P. Bobillier (1980) Retrograde axonal transport following injection of (3H) serotonin in the olfactory bulb. 11. Radioautographic study. Brain Res., 196t417427. Cajal, S.R. (1909-1911)Histologie du systeme nerveux de l’homme et des vertebres. Paris, Maloine. Reprinted Madrid, Consejo Superior de Investigaciones Cientificas, Vol. 1, 1952. Droz, B., A. Rambourg, and H.L. Koenig (1975) The smooth endoplasmic reticulum: Structure and role in the renewal of axonal membrane and synaptic vesicles by fast axonal transport. Brain Res., 93t1-13. Gamrani, H., and A. Calas (1980) Cytochemical, stereological and radioautographic studies of rat raphe neurons. Mikroskopie, 3 6 - 1 1 . Hannah, R.S. (1978) Three-dimensional reconstruction of specialized endoplasmic reticulum in neurons of the rat substantia gelatinosa. Neurosci. Lett., 9:105-111. Markov, D., A. Rambourg, and B. Droz (1976) Smooth endoplasmic reticulum and fast axonal transport of glycoproteins, an electron microscope radioautographic study of thick sections after heavy metals impregnation. Journal de microscopie et de Biologie Cellulaire, 25t57-60. Nissl, F. (1894) Uber des soganannten Granula der Nervenzellen. Neurol. Zentralb, 13t676-685, 781-789. Palay, S.L., and G.E. Palade (1955)The fine structure of neurons. J. Biophysic and Biochem. Cytol., lt69-88. Peters, A,, S.L. Palay, and H. de F. Webster (1976) The Fine Structure of the Nervous System: The Neurons and Supporting Cells. W.B. Saunders, Philadelphia. Rambourg, A,, and B. Droz (1980) Smooth endoplasmic reticulum and axonal transport. J. Neurochem., 351625. Rambourg, A,, Y. Clermont, and A. Beaudet (1983) U1trastructural features of six types of neurons in rat dorsal root ganglia. J. Neurocytol., 12:47-66. Thiery, G . (1979) Colorations signaletiques electives sur coupes epaisses du reticulum endoplasmique, de la chromatine et des surfaces cellulaires libres des cellules animales. Biol. Cellulaire, 35159-164. Thiery, G., and A. Rambourg (1976) A new staining technique for studying thick sections in the electron microscope. Journal de Microscopie et de Biologie Cellulaire, 26t103-106. Tsukita, S., and H. Ishikawa (1976) Three-dimensional distribution of smooth endoplasmic reticulum in myelinated axons. J. Electron Microsc., 25141-149.