THE FINE STRUCTURE OF THE LATERAL VESTIBULAR NUCLEUS IN THE RAT

I. and Neuroglial Cells

CONSTANTINO SOTELO and SANFORD L. PALAY Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 From the Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115. Dr. Sotelo's present address is Laboratoire de Biologie Animale, Facult6 des Sciences de Paris, Paris, France

ABSTRACT The lateral vestibular nucleus consists of multipolar isodendritic neurons of various sizes The distal segments of some display broad expansions packed with slender mito- chondria and glycogen particles. These distinctive formations are interpreted as being growing tips of dendrites, and the suggestion is advanced that they are manifestations of architectonic plasticity in the mature central . Unlike large neurons else- where, the giant cells (Deiters) contain small Nissl bodies interconnected in a dense mesh- work. The Nissl substance is characterized by randomly arranged cisterns of the and by a high proportion of free . Whether attached or free, ribosomes usually cluster in groups of four to six, and larger polysomal arrays are rare. Free ribosomal clusters also occur in the hillock and the initial segment. The neuronal perikarya contain distinctive inclusions consisting of a ball of enveloped by a complex honeycombed membrane. The failure of these fibrillary inclusions to stain with silver sug- gests that the putative argyrophilia of neurofilaments may reside in an inconstant matrix surrounding them. Giant cells of Deiters are in intimate contact with two kinds of cellular elements-astroglial processes and synaptic terminals. Oligodendroglial cells are only rarely satellites of giant cells; in contrast, they are frequently satellites of small and medium- sized cells.

INTRODUCTION

The lateral vestibular nucleus has recently at- and their distribution? This nucleus receives tracted a great deal of attention from investigators important afferent fibers from the vestibular concerned with integrative activity in the central apparatus, the , the reticular formation, nervous system and with chemical changes asso- and the , and it gives rise to a massive ciated with nervous function. In view of this efferent bundle that ends in the spinal cord. The interest, it is important for neurobiologists to have nucleus thus contributes strategically to the control a clear idea of how the nucleus is organized: what of muscular coordination and the maintenance are the characteristics of its constituent neurons, of equilibrium. The variety of afferents in the what is the relationship between the neurons and nucleus offers the cytologist an opportunity to the supportive neuroglial cells, and what are the study the different characteristics of nerve ter- distinguishing features of the various afferents minals from many sources. As the nucleus contains

151 both extremely large nerve cells (the giant cells of The best results were obtained with the method of Deiters) and smaller cells, it also offers an op- double perfusion with aldehydes and osmium tetrox- portunity to analyze the connections made with ide. All the electron micrographs shown in this paper different kinds of nerve cells. were taken from material fixed in this way, except for Fig. 22, which was derived from tissue perfused Much is already known about the topographic with aldehydes alone. organization of the nucleus in terms of the distribu- Thin slices of the brain stem were cut in the coronal tion of afferent nerve fibers, but knowledge of the plane and postfixed in fresh cold 2% osmium tetrox- interrelations between neurons and neuroglial ide solution for 2-3 hr. During this period, the lateral cells, or between these cells and the different kinds vestibular nuclei in the slices were identified under of nerve terminals, is fragmentary. In the present the dissecting microscope and trimmed. After com- work, we have attempted to understand the plete dehydration in ascending concentrations of organization of the lateral vestibular nucleus at methanol, tissue blocks were embedded in Epon 812 the fine structural level. By means of optical and or in Araldite. After polymerization, ultrathin sec- tions were made with a glass knife on a Porter-Blum electron microscopy as well as experimental MT-2 microtome. These sections were mounted on neuroanatomical techniques, we have made a

copper grids, with or without a Formvar-carbon Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 cells and nerve fibers. correlated analysis of the coat, and were double-stained with a saturated Our results will be presented in a series of papers aqueous solution of uranyl acetate followed by lead of which this is the first. Here we present the citrate (40). Sections were examined in an RCA EMU cytology of the nucleus, the fine structure of its 3G electron microscope. Thicker sections (1-2 ,) for constituent neurons and neuroglial cells, and their light microscopy were also cut from the same blocks processes. In later communications, we shall and were stained with toluidine blue. describe the morphology of the terminals in the Blocks of tissue obtained from the brain stems of nucleus and alterations in them after experimental other rats were prepared for silver by Cajal's neurofibrillary method (31). Additional silver prepa- interruption of the afferent pathways. rations were made according to a slight variation of MATERIALS AND METIIODS the Golgi-Rio Hortega method (34) as follows. Several adult rats were perfused with the modified Karnovsky 1 The brains of 21 normal female rats, weighing aldehyde solution, followed by a freshly prepared from 150 to 300 g, were fixed by perfusion through aqueous solution of 6% potassium dichromate, 6o the ascending aorta according to a method previously chloral hydrate, and 4% formaldehyde. After the described (27). Each animal was anesthetized by an perfusion, brain stem slices 3 mm thick and containing intraperitoneal injection of 35 mg of chloral hydrate the lateral vestibular nucleus were postfixed by per 100 g body weight. Four rats were perfused with immersion in the same Golgi-Rio Hortega fixative warm balanced salt solution (18) followed by 2, for 48 hr, with one change of fixative after the first Os0 4 containing 0.5 g calcium chloride per 100 ml 24 hr. The slices were impregnated in 1.5% silver of solution and either 1% polyvinylpyrrolidone or nitrate for 3 days. Sections 80-100 u thick were cut 6% dextran, buffered with 0.07 M acetate-veronal with a freezing microtome and after dehydration (pH 7.1-7.4). Thirteen rats were perfused first with were mounted in Permount under cover glasses. 100 150 ml of a warm solution containing 1% para- formaldehyde, 1.25%, glutaraldehyde, and 0.05c, OBSERVATIONS calcium chloride, and buffered with 0.07 M sodium cacodylate (pIt 7.2 7.4) [modified from Karnovsky Situated in the ventral lateral wall of the IVth (16)]. This solution was followed by 200 ml of 2~, ventricle, the lateral vestibular nucleus occupies a Os04 containing 0.5/`6 calcium chloride and buffered broad zone that is limited rostrally by the superior with 0.1 M sodium cacodylate. The four remaining vestibular nucleus, caudually by the descending rats were perfused with about 500 ml of a warm solu- vestibular nucleus, laterally by the inferior cerebel- tion of 0.12 M phosphate buffer (pH 7.4) containing lar peduncle, and medially by the medial vestib- the following compounds in each 100 ml: paraformal- ular nucleus. The ventral boundary of this nucleus dehyde, I g; 25%/, glutaraldehyde, 4 ml; calcium is formed by the trigeminal complex and the chloride, 2 mg. Fixation in this solution was pro- longed for several hours, and blocks taken from these reticular formation, while dorsally the nucleus brains were postfixed by immersion in osmium tetrox- extends along the medial margin of the inferior ide as described below. cerebellar peduncle into the cerebellum. Although the nucleus is readily demarcated in sections, the 1 Obtained from Charles River Breeding Labora- constituent neuronal cell bodies are not compactly tories, North Wilmington, Massachusetts. aggregated, but are dispersed into small groups of

152 THE JOURNA. OF CELL BIOLOGY · VOLUME 36, 1968 three or more cells interlaced with and distances in all directions throughout the nucleus; numerous heavily myelinated fibers. Perikarya of for example, dendrites of neurons in the dorsal neuroglial cells, both and oligodendro- part of the nucleus reach as far as the ventral cytes, are relatively sparse. part and dendrites originating in the ventral part The neurons are usually multipolar. They can be reach into the dorsal part. Dendritic thorns or classified into three types: the giant cells of Deiters, spines are infrequent, even on the terminal medium-sized neurons, and small neurons. In the branches. rat, as in the cat (3), the caudal part of the nucleus As the cytology of the neurons in the lateral contains more giant cells than does the rostral vestibular nucleus resembles that of neurons part. In the nucleus as a whole, however, medium- generally, the following description will emphasize sized and small neurons are more numerous than only those features that seem to be peculiar to the giant cells that are considered to be typical of this nucleus or that have not been described before, it. even though generally present in other neurons. The neurons of this nucleus belong to the category designated as "isodendritic" by Ram6n- The Giant Cells of Deiters Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 Moliner and Nauta (29), and all of the cells have In these cells, the nucleus is usually large, a similar configuration regardless of size (Fig. 1). spherical, and excentrically placed. Its contour is They are elongated polyhedrons with long, fairly smooth and free of the indentations and folds that straight dendrites. These give rise to relatively mark the nuclei of some large neurons in other few branches, which leave at acute angles to the regions of the nervous system. The nuclear enve- thick main stem. The dendrites extend for long lope has a gently undulating profile only rarely

FIGURE 1 Light micrograph of a Golgi-Rio Hortega preparation of the lateral vestibular nucleus in an adult rat. X 280.

C. SOTELO AND S. L. PALAY The Lateral Vestibular Nucleus. 1 153 Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021

FIGURE Electron micrograph of the cytoplasm of a giant cell of Deiters, showing the small masses of Nissl substance (N) interconnected to form a coarse network. In the remaining cytoplasm, containing mitochondria and the Golgi apparatus (G), neurofilaments and microtubules are disposed in arcs around the Nissl bodies. X 10,000. studded with attached ribosomes. The nuclear small number of ribosomes in the polysomes of the content consists of fine filaments, disposed singly or giant cells in the rabbit has recently been pointed in pairs, and associated granules varying in size out by Ekholm and Hyd6n (7). from that of ribosomes to twice as large. Although The cytoplasm between the Nissl bodies con- most of the nuclear chromatin is rather uniformly tains the usual organelles: Golgi apparatus, dispersed, small blocks of it occur in the form of mitochondria, lysosomes, microtubules, neuro- coiled strands densely aggregated against the filaments, pigment granules, and vesicles of various nuclear envelope. The nucleolus, as in most sizes. The Golgi apparatus is clearly displayed neurons, forms a prominent globular body lying surrounding the nucleus and extending into the off-center in the nucleus. It consists of coarse, largest dendrites. As can be seen in Figs. 3 and 8, dense granules compacted into a thread that is the flattened cisterns of the Golgi apparatus are loosely coiled upon itself. Open spaces between the associated with numerous vesicles and clumps of turns of this nucleolonema can be large enough to multivesicular bodies. Many of the vesicles in the be discernible in the optical microscope as round, Golgi region are "coated" or "alveolate," and pale inclusions within the nucleolus. many of the smaller ones contain a dense spherical Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 In preparations stained for optical micropcsoy, inclusion about 500 A in diameter. The significance the cytoplasm of the giant cells is characterized by of these inclusions is not clear. large Nissl bodies similar to those in motoneurons. The mitochondria are generally small, varying In electron micrographs of thin sections, however, from 0.1 to 1.5 in diameter, and are elongated in the masses of Nissl substance are small. They are form. Their ultrastructure is similar to that ob- interconnected by fine strands of the same compo- served in other neurons. Occasionally, a mito- sition into a coarse network that is dispersed chondrion in the peripheral cytoplasm of the throughout the cytoplasm except for a narrow is associated with a subsurface cistern, as band beneath the surface of the neuron. The has been reported by Rosenbluth (35) in spinal discrepancy between these two appearances is cord neurons and by Herndon (12) in Purkinje probably explained by the thickness of the section cells. Although this pairing of a mitochondrion used for optical microscopy, which permits many with a subsurface cistern often occurs beneath an nodal points in the network to overlap within the attached to the cell membrane of image plane. The fluffy texture of the Nissl bodies the neuron (Fig. 5), this relationship is by no in the thick sections is probably the counterpart means exclusive. This association of organelles of the reticular dispersion of the Nissl substance also occurs beneath the neuroglial investment of as seen in thin sections. The cisterns in the endo- the cell. It should be pointed out here that, since plasmic reticulum of the Nissl substance are often the giant cells of Deiters are covered only by disposed in parallel array (Fig. 6), but commonly synaptic terminals and astroglial processes (see they are randomly arranged, with numerous page 166), the apparent association of these branchings or anastomoses between neighbors striking organelles with either axon terminals or (Fig. 2). This arrangement conforms to the usual neuroglia is merely a matter of chance. pattern in the small Nissl bodies of most neurons, The cytop]asmic matrix of the giant cells is and it is particularly well shown in the strands of occupied by numerous microtubules and neuro- Nissl substance interconnecting the larger masses. filaments that are disposed in arcs swirling and In between the cisterns of the endoplasmic streaming around the more massive Nissl bodies reticulum, large numbers of ribosomes are sus- and mitochondria. A certain amount of order is pended in clusters of four to six granules. Only a discernible in these zones. The filaments and small proportion of the ribosomes are attached to tubules often run parallel with one another, form- the outer surfaces of the cisterns, and most of the ing arrays in which the strands are equidistant. cisterns are free of ribosomes. Therefore, the endo- Sometimes a short length of tubule or filament plasmic reticulum in these nerve cells can be con- lies athwart the main stream of parallel elements, sidered as generally smooth (or agranular), even indicating that some of them undergo gradual though the surrounding matrix abounds in ribo- twisting or spiraling. In the zones occupied by these somes. The polysomal arrays attached to the structures there are also very fine granules, thin membranes consist of straight chains or short weblike strands, and pale filaments, which prob- spirals of four to six ribosomes. The relatively ably are all images of the same structure captured

C. SOTELO AND S. L. PALAY The Lateral Vestibular Nucleus. 1 155 in different planes of section. The granules have does not stain, it is easily overlooked or rejected less than one-third the diameter of a and as an artifactitious vacuole. In electron micro- are much less dense. Their size, pallor, and fuzzy graphs of thin sections, it appears in its simplest outlines make them difficult to measure with any form (Fig. 9) as a circular area filled with long, precision. Many granules and strands adhere to slender threads about 100 A in diameter, to which the microtubules and neurofilaments (Fig. 20), delicate, fluffy material adheres at regular inter- producing a semblance of periodicity, but it is vals. The filaments run roughly parallel with one unclear whether these subsidiary structures should another and are set apart by equal distances be considered as intrinsic to the filaments and like the neurofilaments in the rest of the cytoplasm microtubules or as merely a kind of precipitate and in . In these inclusions, however, the upon them. filaments are arranged in bundles gently coiled o, The number of microtubules in a given field of braided about one another (Figs. 9, 13, 14, and 17). cytoplasm is inversely proportional to the number In three dimensions the inclusions would appear of neurofilaments. In some neurons microtubules as if filled with a knot of coarse twisted twine. As

predominate, whereas in others the neurofilaments these inclusions contain a highly concentrated and Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 predominate. Although these differences between almost pure population of neurofilaments, they neurons can indicate different functional states, it offer an unusual opportunity to test the argyro- must also be noted that there are considerable philia of these filaments. In sections prepared ac- differences from one field to another within the cording to Cajal's reduced silver method, however, same cell. For example, Fig. 3 shows the perinu- the inclusions remained recognizable but un- clear zone of a giant neuron with numerous neuro- stained, although neurofibrillae in the neurons and filaments coursing among the Nissl bodies and their processes were intensely impregnated Golgi apparatus. A similar region of a cell shown (Fig. 10). in Fig. 4 contains a centriole in addition to the Some of the inclusions contain a dense core con- other organelles, and in this micrograph there are sisting of closely packed filaments (Figs. 11, 14, numerous microtubules concentrated at the right and 19). In these regions, the filaments are so ir- where they radiate from a central point. This is regularly interlaced that it is impossible to be sure probably the edge of the centrosphere associated that all of the punctate images represent transverse with the partner of the centriole in the middle of sections of filaments rather than granules. Another the picture. Thus, local variations in the distribu- kind of filamentous material occurs rarely in the tion of cell organelles can give rise to misleading inclusion. This consists (Fig. 14) of threads radiat- impressions of the balance between neurofilaments ing from a dense, finely granular central mass, and and microtubules in a given cell. the threads themselves appear as rows of alternat- Besides being dispersed throughout the cyto- ing densities, suggesting a helix. Not enough exam- plasmic matrix, the neurofilaments participate in ples of this material have been encountered to the formation of a peculiar inclusion that occurs permit us to give a more precise description. Other commonly in the giant cells of Deiters and is here rare components of the inclusion are small numbers described for the first time. When once discovered of clustered ribosomes (Figs. 14, 15, and 19) and in electron micrographs, this inclusion was readily small vesicles with either clear or dense centers recognized in 1-/g sections stained with toluidine (Figs. 9 and 14). blue and examined in the optical microscope. In These fibrillary inclusions are delimited from the the latter preparations (Fig. 12), it appears in the cytoplasmic matrix by a wall of varying com- cytoplasm as a round or oval clear area sometimes plexity. In the simplest forms, the wall is a continu- containing a dense central spot. As the inclusion ous single membrane about 70 A thick (Fig. 9), but

FIGURE 3 Electron micrograph of the perinuclear region of a giant cell of Deiters, showing the Golgi apparatus (G). Granular vesicles and alveolate vesicles (arrows) are numerous in this region X 15,000.

FIGURE 4 Electron micrograph of the cytoplasm of a giant cell showing the Golgi ap- paratus (G) and a centriole (c). At the right, the numerous microtubules are probably part of the centrosphere (s) associated with the partner of this centriole. X 13,600.

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C. SOTELO ANI) S. L. PAIAY The Lateral Vestibular Nucleus. 1 157 in many regions it consists of appressed tubular Occasionally, three types of peculiar nuclear structures that give it a multiloculated, honey- inclusions have been encountered: (1) bundles of combed appearance (Figs. 11, 14, and 16-19). long, thin filaments, similar to those described by Some inclusions are surrounded completely by one Gray and Guillery (9) in cells of the leech or more such honeycombed envelopes (Figs. 14, 16, and by Siegesmund et al. (37) in the granule cells and 18). The limiting membrane does not appear of mammalian olfactory bulb and cerebellum; (2) to be continuous with either the rough or smooth thin sheets of fine parallel filaments arrayed in a endoplasmic reticulum in the surrounding cyto- lattice, similar to those reported by Chandler and plasm. Willis (4) in neurons of cerebral cortex, thalamus, The inclusions vary in diameter from 0.2 to 6 u. and superior colliculus; and (3) bouquets of long, Several are frequently encountered in any one sec- parallel microtubules about 270 A in diameter, as tion through a cell, and they can be distributed as shown in Fig. 24. The first two types have also been isolated individuals anywhere in the perikaryon. seen in the nuclei of cultured Usually, however, they tend to be clustered to- cells subjected to X-irradiation (17). The third

gether, interconnected through peduncles formed type of inclusion has not been reported before. As Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 by their complex envelope. In such instances, the the microtubules in it are associated with fine fila- individual inclusions often lose their spherical ments, this inclusion may be related to the other shape, becoming elongated and irregular, as if two. It is not clear whether any of the inclusions compressed together (Fig. 11). Even isolated in- correspond to Cajal's "batonnets intranucleaires" clusions have small satellites of the same composi- (30), as Siegesmund et al. have suggested. In elec- tion attached to, or included in, their envelopes tron micrographs they appear to be very rare, (Fig. 17). whereas Cajal's comments indicate that the baton- nets are common. Only one example of these nu- Neurons of Medium and Small Size clear inclusions has been observed in the giant As these neurons resemble the giant cells of cells-a bouquet of microtubules and fine filaments Deiters in most particulars, the following remarks corresponding to the third type. As the incidence will be devoted to observations of differences be- of the inclusions, even in the smaller cells, is not tween the two types of cells. One of the most im- large, they may have been missed. pressive differences, one that is visible even in the In the cytoplasm of the small and medium-sized optical microscope, is that the nuclei of the small neurons, the Nissl bodies are small and widely and medium-sized cells occupy a much larger dispersed throughout the perikaryon. In contrast proportion of the cross-sectional area of the cell to the characteristic pattern in the giant cells, body. The nuclear envelope is deeply invaginated Nissl bodies frequently appear in the most by cytoplasm filled with clustered ribosomes and a peripheral cytoplasm (Fig. 23), where they under- few vesicles (Fig. 23). The circular profiles of the lie either synaptic terminals or neuroglial processes folds in transverse section show that they are adjacent to the surface of the neuron. usually long, finger-shaped indentations, rather Although the association of subsurface cisterns than broad wrinkles in the surface of the nucleus. and mitochondria is rarer in the smaller cells than

FmuIGE 5 Peripheral region of a giant cell, showing the association of a mitochondrion with a subsurface cistern (arrow). X 25,000.

FIGUuRE 6 Nissl body of a giant cell with the endoplasmic reticulum disposed in parallel array. Most of the ribosomes are free, arranged as small polysomal clusters. X 26,000.

FIGURE 7 Oblique section through a cilium (cil) of a medium-sized neuron. X 26,000.

FIGuRE 8 The most peripheral region of a giant cell is devoid of Nissl substance, being occupied by free ribosomes, mitochondria, and arrays of parallel neurofilaments and micro- tubules. The Golgi apparatus (G) is often associated with clusters of multivesicular bodies (my). X 13,000.

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C. SOTELO AND S. L. PALAY The Lateral Vestibular Nucleus. I 159 in the giant cells, stacks of several closely apposed encountered initial segments in transverse section, cisterns beneath the surface are more frequent. a failure that is probably merely fortuitous, for we These groups of cisterns, with the last member have found them in oblique sections. studded with ribosomes (Fig. 25), resemble those The and initial segment character- described by Rosenbluth (35) in the pyramidal istically contain sheaves of parallel microtubules cells of the cerebral cortex. which collect in the broad portion of the hillock Centrioles and cilia (Fig. 7) are encountered (Fig. 22) and extend into the axon until the begin- more frequently in the smaller cells than in the ning of the sheath. Here they either termi- giant ones. In transverse section (Fig. 26), it can be nate abruptly or disperse. The microtubules ap- seen that the cilium contains eight doublets in its pear to be bound together by dense material periphery and one off-center, as described by Dahl distributed like bridges at irregular intervals along (5). Isolated fibrillary inclusions, like those found their length. The close association of these tubules in the giant cells, also occur in the cytoplasm of into bundles or ribbons produces, in longitudinal small and medium-sized neurons. sections, an appearance of extreme density due to the fact that two or three microtubules overlap Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 Axon Hillock within the thickness of the section. The bundles, Fig. 21 shows a longitudinal section through the consisting of three to six microtubules, are dis- axon hillock and initial segment of the axon be- tributed across the axon without any definite pat- longing to a giant cell of Deiters. The axon leaves tern. Although individual microtubules could be the perikaryon by a funnel-shaped process, nar- followed for 2-3 p along the length of the axon, we rows to a slender fiber about 2 A across and about found none that shifted from one bundle to an- 27 u long, which is coated by thin neuroglial proc- other. An extensive series of sections in the trans- esses, and then enters the first segment of its myelin verse plane would be necessary for one to ascertain sheath. The internal structure of this axon is whether a particular microtubule always remains typical of the initial segments of both giant cells with its primary bundle. and smaller cells, and they conform to the pattern Aside from the microtubules, the initial segment seen in other multipolar neurons (28). In this contains many elongated mitochondria generally study, we have found the axon hillock of 30 differ- arranged with their long axes parallel to the axon ent cells having the axon continuous with the and especially congregated in the axon hillock perikaryon, and in two of these the entire length region (Fig. 22). There are also many neurofila- of the initial segment from hillock to myelin sheath ments and tubules of endoplasmic reticulum, was included in the micrographs. In one case, we which are drawn out longitudinally, parallel with have found the point of emergence of the axon the other structures. from a large , as Cajal (30) has described Contrary to what might be expected from prepa- in Golgi preparations. We have not, however, rations stained with basic dyes, the axon hillock is

FIGmUE 9 Simplest form of the fibrillary inclusions in the neurons of the lateral vestibular nucleus. The arrow points to a part of their wall formed by only a single membrane. A cluster of ribosomes (r) is found in one of these bodies. X 13,000.

FIGURE 10 Light micrograph of a giant cell of Deiters stained according to the Cajal neurofibrillary method. Neurofibrils in the perikaryon and dendrites are well impregnated. The arrow points to the negative image of a fibrillary body, which has not been impreg- nated. X 900.

FIGURE 11 Large aggregate of fibrillary inclusions in a giant cell. The inclusions have lost their spherical shape, but retain their individuality, each one being surrounded by a wall. Some of the inclusions contain a dense core (D) formed by closely packed filaments. X 33,000.

FIGURE 12 Light micrograph of a giant cell stained with toluidine blue. Three inclusions (arrows) appear as unstained vacuoles; two of them contain a dense central spot. X 1,500.

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FIGURE 13 Peripheral region of a Deiters cell showing two fibrillary bodies (F) and one axon terminal deeply invaginated into the cytoplasm and suggesting an intracellular axon terminal. X 19,000.

not devoid of ribosomes. It is true that large aggre- boutons are attached to the initial segment, gen- gates and complete Nissl bodies do not encroach erally near its origin from the cell body. The upon the hillock (Fig. 22), yielding a relatively undercoating of punctate axoplasmic material is clear zone (occupied by mitochondria, neurofila- absent under the part of the membrane that lies ments, and microtubules) from which the axon opposite these synaptic terminals. arises. But small clusters of ribosomes, usually in polysomal array, do extend into the hillock and the The Neuropil initial segment, becoming more and more sparse The greater part of the nucleus is occupied by until the region of the myelin sheath is reached myelinated fibers, some of which approach close where they disappear altogether (28). The ribo- to the cell bodies and give off slender preterminal somes are not usually attached to the membranes unmyelinated branches or end abruptly as large of the endoplasmic reticulum. bulbous terminals. Many of the myelinated fibers. The surface of the axon in its initial segment is however, are fibers of passage on their way to the specialized by the presence of an undercoating of cerebellum and to the other vestibular nuclei. fine granules. The coating first appears at the Among these fibers and neuronal perikarya are point where the axon hillock narrows to form the small islands of neuropil consisting of dendrites, beginning of the initial segment and disappears unmyelinated with their associated synaptic when the axon enters the myelin sheath. Occa- terminals, and neuroglial cells. As in other regions sionally, alveolate vesicles or pits occur in the of the mammalian , the ex- surface of the initial segment, as well as small tracellular space is represented by an interval of thornlike projections. Small presynaptic terminal about 150 A between the limiting membranes

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FIGURE 14 Complex fibrillary bodies displaying several kinds of filamentary arrangments. The arrow points to filamentous material forming threads radiating from a dense central mass. In one of these in- clusions, clusters of ribosomes (r) are present. This micrograph also shows the tubular structure of the wall of the fibrillary inclusions. X 7,000.

of the cellular elements that compose the neuro- tion, they can give the impression of continuity. pil. Fig. 20 shows several points of overlap, and the As the on the surfaces of the perikarya arrow indicates one in which the boundary sepa- and dendrites vary considerably in their form and rating the microtubule from the endoplasmic complexity, the full description of them will be re- reticulum is clear. This interpretation has already served for the second paper of this series. In the been advanced by Metuzals (19) in a study of present paper, we shall confine our attention to the axons in the diencephalon of the frog. structure of dendrites and axons coursing through The dendrites of the lateral vestibular nucleus the neuropil. Fig. 20 shows a longitudinal section display certain peculiar features of general interest. of a dendrite in which microtubules, neurofila- Longitudinal sections (Fig. 32) show that some of ments, and agranular endoplasmic reticulum can the dendrites suddenly enlarge to form varicosities be distinguished. The endoplasmic reticulum oc- filled with mitochondria. The swellings vary from curs in a tubular form whose irregular swellings 1 to 8 / in diameter, and some are even larger. In deceptively appear to be continuous with some of nearly every instance, their dendritic nature is the microtubules. This appearance is fairly fre- confirmed by the presence of axonal terminals quent in longitudinal sections of dendrites and has synapsing upon them (Figs. 28, 30, and 31). In given rise to some confusion (24). Two different addition, a few leaflike or club-shaped processes longitudinal and tubular structures are involved are given off from these varicosities to make synap- here. When they overlap in the thickness of a sec- tic contact with axonal terminals in the surround-

C. SOTELO AND S. L. PALAY The Lateral, Vestibular Nucleus. 1 163 ing neuropil (Figs. 31 and 32). The mitochondria particles within deep, cup-shaped depressions in in these dendritic varicosities are long and slender the surface of the organelles. Three such distorted (0.2-0.25 /j in diameter) and generally contain mitochondria are shown in Figs. 29 and 30, where longitudinally oriented cristae. They either lie possible stages in their formation can be seen. In parallel to the long axis of the dendrite or form a Fig. 29, mitochondrion I displays an armlike ex- gently swirling figure around the long axis (Fig. tension partially surrounding a few glycogen par- 28). Usually, transverse sections show that the ticles; mitochondrion 2 completely encloses a mitochondria are remarkably uniform and each is comparable cluster. These extensions appear to in almost perfect alignment with its neighbors consist of the inner and outer membranes of the (Figs. 29 and 30). The number of mitochondria mitochondrial envelope and some included matrix, that can be packed into such profiles is astonishing. but no cristae. In Fig. 30, a field of glycogen par- In one instance, 134 mitochondria were counted ticles is surrounded by an incomplete ring formed inside a dendrite of 12.5 2 cross-sectional area, and by the mitochondrial envelope and the enclosed in another instance 297 mitochondria were matrix. These figures may be precursors of other 2 crowded inside a dendrite of 26 u. forms, more difficult to interpret with certainty, in Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 Despite this crowding, there is room in the den- which double-walled vacuoles containing mem- drite for other organelles: microtubules running branous laminae resembling mitochondrial cristae longitudinally, agranular endoplasmic reticulum appear with or without enclosed glycogen particles. in the form of meandering tubules and small cis- All of these figures can be distinguished from arti- terns, multivesicular bodies, and various types of factitious swellings or explosions of mitochondria, vesicles. Aside from the mitochondria, the most such as are associated with glutaraldehyde fixation. striking inclusions in these dendritic profiles are In the latter forms, the extensions from the mito- glycogen particles, which vary from single, irregu- chondrial envelope involve only its outer mem- lar granules ( particles), 200-300 A in diameter, brane. to rough clumps (a particles), 800-1000 A in diam- eter and consisting of four to eight individual 3 Neuroglia particles (Fig. 29). In most instances, glycogen Two types of neuroglial cells, astrocytes and particles are dispersed irregularly among the mito- , occur in the normal lateral ves- chondria, but sometimes glycogen fills so much of tibular nucleus. Since theultrastructure of these cells the profile that the mitochondria are displaced to is not unusual (22, 26, and 27), we shall devote our the periphery, where they remain curiously attention to the relations between neurons and crowded together (Fig. 31). neuroglial cells. Frequently, mitochondria sequester glycogen A large proportion of the oligodendrocytes are

FIGURE 15 Clusters of ribosomes (r) in a fibrillary body. X 16,000.

FIGURE 16 Tangential section through the wall of the fibrillary inclusions showing its honeycombed appearance. X 31,000.

FIGURE 17 Isolated fibrillary body with a small satellite included in its envelope. X 19,000.

FIGURE 18 Multiloculated appearance of the envelope of several small fibrillary inclu- sions. X 25,000.

FIGURE 19 Large complex fibrillary bodies, one with a dense core (D) of closely packed filaments, and another with clusters of ribosomes (r). X 25,000.

FIGnRE 20 Longitudinal section of a dendrite showing neurofilaments (nf), micro- tubules (mt), and agranular endoplasmic reticulum in tubular form (er). Some points of overlap between microtubules and endoplasmic reticulum can be seen; in one of them (arrow) the boundary separating the two structures is evident. X 40,000.

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C. SOTELO AND S. L. PALAY The Lateral Vestibular Nucleus. 1 165 found in those areas of the nucleus that are oc- (Fig. 27) that resemble immature myelin. Al- cupied almost exclusively by myelinated fibers. In though the neuronal perikarya are usually sepa- the neuropil, extensions of the oligodendrocytes rated by zones of neuropil, or at least by a thin are rarely encountered. Furthermore, these neuro- lamella of astrocytic cytoplasm, there are occa- glial cells are not usually found in immediate ap- sional instances in which one neuron abuts directly position to a giant neuron in the lateral vestibular upon another with only the usual interstice of nucleus. In the rare instances in which an oligo- 150 A between them. In these regions of immediate dendrocyte appears to be a satellite of a giant, or contact, which can extend over several micra, the Deiters, cell, there is almost always a thin layer of adjacent surface membranes display no specialized astrocytic cytoplasm interposed between the oligo- junctional characteristics. dendrocyte and the neuronal cell body (see Fig. 22). Most of the surface of the giant cell is covered DISCUSSION by synaptic boutons, and the remainder is covered The advantage of an electron microscopic analysis by slender astrocytic expansions and myelinated of a small region in the brain is that it reveals de- fibers. Generally, the oligodendrocytes in the Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 tails of structure and intercellular relations that neighborhood of a giant cell are located at a little are not discernible in optical microscopic studies. distance from the neuron and are separated from it Electron microscopy does not replace optical by a narrow zone containing synaptic boutons microscopy and its time-honored techniques, but enveloped by lamellae of astrocytic cytoplasm. extends the range and precision of the observer and Most of the perineuronal oligodendrocytes are limits the number of possible interpretations. It satellites of the small or medium-sized neurons. may be anticipated that an electron microscopic One of these is shown in Fig. 23, where an oligo- study of a specific region will disclose structural dendrocyte can be seen in close association with features that, upon further study, prove to be gen- part of the neuronal surface. The rest of the surface erally applicable to many other regions. Other is covered by myelinated nerve fibers or lamellar features, however, may prove to be restricted to expansions of astrocytes. the region or cell under study and may, therefore, The common neuroglial cell of the neuropil is provide valuable clues to its special function or the protoplasmic (Fig. 27), which sends organization. Because of these encouraging possi- out numerous fine lamellar processes extending between the other cellular elements. The lamellae bilities, neuroanatomists are stimulated to continue enfold dendrites and axons and insinuate them- their cytological exploration of the central nervous selves into the crevices around the neuronal peri- system at the fine structural level until the general karya, leaving small apertures for the entrance of pattern is well filled out. the preterminal axons that upon them. As In the present paper, we have reported many a result, nearly all neuronal surfaces are coated by similarities between the cells of the lateral vestibu- one or more layers of astrocytic cytoplasm, except lar nucleus and those of other parts of the central where synaptic contacts are made. Sometimes the nervous system. It was not to be expected that astrocytic lamellae form spirally wound figures these cells should differ in their essential structure

FIGURE 21 Origin of the axon of a giant cell. The initial segment, about 27 long and 2 across, extends from the axon hillock on the left to the beginning of the myelin sheath on the right. The sheaves of parallel microtubules within the initial segment disappear when the axon enters the first segment of its myelin sheath. X 6,000.

FIGURE 22 Axon hillock of a giant cell of Deiters. Clusters of free ribosomes (r) are numerous in this region, but Nissl bodies (N) are absent. The consequent pallor of the transitional zone between the perikaryon and the initial segment of the axon is evident in this electron micrograph as well as in photomicrographs. The arrow points to a thin layer of astrocytic cytoplasm located between the neuron and a neighboring (0). X 10,000.

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FIGURE 23 Medium-sized neuron. The nucleus shows some finger-shaped indentations. One oligodendro- cyte (0) is in a satellite position to this neuron. X 14,000. 168 Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021

FIGURE 24 Nuclear inclusion, in a medium-sized neuron, formed by a bundle of microtubules associated with fine filaments. X 26,000.

FIGURE 25 The arrow points to a stack of three closely apposed cisterns beneath the surface of a small neuron. X 20,000. from the general picture of neurons and neuroglia with a row of two or three inclusions leading to an already well established by earlier studies. Never- apparently intracellular axonal terminal, rein- theless, the present report discloses new features of forced this suggestion. But further study revealed nerve cells and their processes and, interestingly, the more complex profiles (Figs. 14, 16, 19), which these features, although not previously described, are very difficult to reconcile with the form of an turn out to be present in other regions of the ner- axon. Furthermore, examination of serial sections vous system as well. in the light microscope disclosed that the inclusions are spherioidal or lobulated, are never attached to The FibrillaryInclusions the surface of the cell, and always begin and end One of these structures is the fibrillary inclusion within the neuron. Occasional images like Fig. 13 body found in both giant and smaller neurons. must be interpreted as accidental coincidences of a Although the nature of this inclusion is obscure, synaptic terminal located in a depression in the certain suggestions about its significance can be neuronal surface and neighboring fibrillary inclu- excluded by our observations. The form of the sions. In addition to the neurofilaments, the inclu- simplest inclusions and their impressive content of sions also contain ribosomes (Figs. 14, 15, and 19) neurofilaments immediately suggested that the in- and dense aggregates of finer, perhaps helical fila- clusions were axons that penetrated deeply into the ments, both of which have not been found in perikaryon. An occasional micrograph like Fig. 13, terminal parts of axons. Moreover, if these were

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FIGURE 26 Parts of two adjacent medium-sized neurons. In the neuropil the arrow points to a transverse section of a cilium containing eight doublets in its periphery and one off-center. X 17,000. axons, their profiles should be surrounded by two have functions to be ascribed to them. The role of surface membranes, the plasmalemma of the axon these structures awaits a more detailed under- and the plasmalemma of the perikaryon. But the standing of the integrated function of the nerve cell limiting envelope of the inclusions does not re- than is now available. semble the plasmalemma. It varies from a single According to investigations by Gray, Guillery, membrane to a complicated honeycomb structure. and Boycott (2, 8), the neurofibrillae shown by All of these features make it clear that they are not optical microscopy in nerve cells, processes, and axons or parts of other cells invaginating the peri- endings correspond to bundles of the neurofila- karyon, but are truly intracellular inclusions. ments shown by electron microscopy. These These inclusions are not specific to the neurons authors have presented strong evidence for the of the lateral vestibular nucleus. They have been conclusion that the neurofilaments are responsible seen in the ventral cochlear nucleus, in the spinal for the argyrophilia of neural tissue. It is, therefore, cord2 , and in the posterior colliculus3. So far, they of some interest to examine the fibrillary inclusions have not been found in any species other than the described in the present paper in the light of the rat. Like many other neuronal inclusions that have conclusions offered by Gray and his coworkers. been described (20, 21, and 39), the fibrillary Although filled with filaments morphologically bodies are mysterious. We now possess a much identical to neurofilaments elsewhere, these fibril- larger catalogue of cytoplasmic organelles than we lary bodies failed to stain with Cajal's neurofibril- lary silver stain. If, on the one hand, the filaments 2 R. B. Wuerker. Personal communication. in these bodies are chemically identical with neuro- 3 D. K. Morest. Personal communication. filaments elsewhere, then we must conclude that

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FIGURE 27 Electron micrograph of an astrocyte containing bundles of fine filaments (f) and giving off seven thin processes within the field. At the left of center, a spiral lamellar arrangment of thin astroplasmic sheaths resembles immature myelin. X 26,000. argyrophilia is not inherent in the filaments them- of the filament itself and may not be resolvable by selves, but in a matrix that surrounds them. In that our methods, or it may not have been preserved. case, the matrix in the inclusions must differ from that around neurofilaments elsewhere. If, on the The Terminal Segments of Dendrites other hand, these filaments are chemically different One of the impressive findings in this study of the from other neurofilaments as shown by their lack lateral vestibular nucleus is the peculiar dendritic of argyrophilia, then we must conclude that there profiles loaded with mitochondria and glycogen. are different types of neurofilaments and that their These profiles are most abundant in the dorsal part morphology is not sufficient to establish either their of the nucleus, but are not restricted to it. They similarities or chemical differences. If the latter have been seen in other regions of the nervous sys- conclusion is correct, we can no longer assume that tem, for example, in the cerebellar cortex and the all filaments found in nerve cells and having the hypothalamus4 and in the superior cervical gan- morphological characteristics of neurofilaments glion.' They have not, however, been observed so (25) are chemically identical. Furthermore, the frequently in these regions as in the lateral vestibu- chemical characterization of the filamentous pro- lar nucleus. It is impossible to decide on the basis Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 tein extracted from squid axons (6, 36) can no of currently available information whether this longer be generalized to other species or even to difference in frequency is due merely to the small other nerve cells. This line of argument seems to sample examined by electron microscopy or to lack lead in a pessimistic direction. Of course, it is possi- of attention on the part of observers. Within the ble that neurofilaments may all be constructed of lateral vestibular nucleus, however, there is a clear the same protein, but that in different situations its difference in the frequency of these profiles surface may have different distributions of charges between the dorsal and ventral parts of the nu- or exposed radicals so that its reaction to the pro- cleus. Therefore, they must belong to the processes cedures of silver staining would be different. of only certain types of neurons and not to all. Fur- Against this possibility is the great dependence of Lher work is necessary in order to identify the cell the conformation of proteins upon these precise type that gives rise to these dendrites. distributions of charges or exposed radicals. The That these profiles represent sections through morphology of the filament is probably congruent dendrites is proved by images like Fig. 32 showing with its chemical constitution. that they are continuous with typical dendrites. We are thus led back to the first alternative: that The location of axonal terminals in presynaptic the argyrophilia is a property of the matrix sur- relation to their surface (Figs. 28 and 30) is further rounding the neurofilaments. In fixed and sec- evidence in favor of this interpretation. It is less tioned material, most neurofilaments (and micro- easy to ascertain which part of a dendrite is repre- tubules) are surrounded by fuzzy weblike strands sented by these profiles. They often appear as a and granules that may represent part of the matrix. dilation or varicosity in the course of a dendrite But it is unlikely that this is the argyrophilic suddenly crowded with mitochondria that are matrix, for it coats the filaments in the fibrillary bodies as well as those elsewhere in the neuron. The 4 S. L. Palay. Unpublished observations. argyrophilic material may be located in the wall T. H. Williams. Personal communication.

FIGUtRE 28 Dendritic expansion filled with mitochondria. The arrow points to an axon terminal synapsing on this dendrite. X 17,000.

FIGURE 29 Dendritic profile containing cup-shaped mitochondria with enclosed glycogen particles. Mitochondrion 1 partially surrounds a few glycogen particles; mitochondrion 2 completely encloses a similar cluster. X 25,000.

FIGURE 30 Part of a dendritic profile containing a distorted mitochondrion forming the wall of a vesicle with enclosed glycogen particles (arrow). An axon terminal (A) synapses on this dendrite. X 31,000.

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FIGURE 31 Two dendritic profiles with the mitochondria displaced peripherally by large masses of glycogen, which occupy most of the dendritic area. Fine finger-like processes (arrows) project from the sur- face of the lower dendrite and an axon terminal (A) synapses with it on the right. X 3,000. Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021

FIGURE 32 Longitudinal section of a dendritic tip. The dendrite (Den) suddenly enlarges, forming a varicosity filled with mitochondria. Two fingerlike projection extend from the distal parts of the tip (arrows). X 29,000.

nicely aligned parallel to the longitudinal axis. The micrographic appearances with the distal dendritic dilated region is, however, so large that the area branches seen in the optical microscope. Although included in a thin electron microscopic section the high concentration of mitochondria in them does not usually contain a further stretch of the should make them rather easy to identify in thick dendrite supposedly narrowing as it leaves the vari- sections, we have thus far been unable to distin- cosity. This circumstance suggests that many of guish them, even at magnifications of 1500 times, these profiles are actually ends of dendrites, a sug- from collections of presynaptic terminals and gestion that is supported by the appearance of glomeruli. Descriptions of the forms of dendritic small finger- or leaflike projections from their sur- tips in Golgi preparations are strangely elusive. faces (Fig. 32). If not the actual terminations, these Dendrites are described as branching repeatedly profiles must represent at least the distal segments and then fading from sight. Either the tips are not of dendrites. Sections passing longitudinally really recognizable in the optical microscope or through the origin of dendrites from the perikaryon they break up into short, thin strands. This vague never display the dense aggregations of mitochon- picture could be explained by the content of these dria and glycogen seen in these profiles. Further- peculiar profiles, if it is remembered that in the more, the part of the dendrite continuous with Golgi preparations the precipitate of silver salts is these profiles is generally narrow and free of deposited in the cytoplasm of the nerve cell and ribosomes, characteristics of the more distal den- its processes but does not impregnate the mito- dritic branches. chondria (1, 38). Thin lamellae of silver salts It would be interesting to compare these electron around or between the mitochondria could

C. SOTELO AND S. L. PALAY The Lateral Vestibular Nucleus. 1 175 produce the appearance of fraying out at the end degeneration and regeneration of nerve endings in of the dendrite. 6 the normal animal. Both axons and dendrites The spectacular aggregation of mitochondria might engage in such a process, reflecting the con- and glycogen in these dendritic profiles poses the tinued experience of the animal, making and dis- question of their function. What is the significance carding interneuronal connections by the activity of the high concentration of mitochondria in just of both pre- and postsynaptic elements. Although this part of the neuron? Nothing that we know this hypothetical interpretation is tempting to in- about the physiology of dendrites points to an vestigators interested in finding a morphological unusually high requirement for energy or other substrate for the process of learning, it would be products of oxidative phosphorylation. The asso- very difficult to test experimentally. ciation of aggregates of mitochondria and abun- A last possibility is that these profiles are path- dant glycogen in these profiles is reminiscent of the ological. This seems to be unlikely because their cytoplasm of brown fat cells (23), where the mito- included mitochondria appear completely normal, chondrial oxidative activity is uncoupled from and lysosomes are lacking. Furthermore, no ab-

phosphorylation. In this example, the role of the normal neurons or neuroglial cells have ever been Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 mitochondrion in heat production seems evident, observed in these specimens. Finally, the dendritic but no such requirement is yet known in the ner- profiles have been seen in many different parts of vous system. It is possible that the mitochondria the brain and in a large number of animals from are actually inactive and have merely collected at different sources. the tips of the dendrites as a result of protoplasmic streaming in the dendrites. The reciprocal relation Ribosomal Patterns in the Giant Cells between quantities of glycogen and number of Ribosomes in the giant cells of Deiters display mitochondria, as shown in Figs. 28 and 31, may two characteristics that are typical of these cells. indicate different states of dendritic metabolism. First, they are more rarely attached to the mem- These peculiar dendritic profiles introduce a branes of the endoplasmic reticulum than in other puzzling problem that is difficult to investigate large neurons, for example, the or the because of their small size and diffuse distribution . Second, the ribosomes are arrayed in a complex tissue. in a distinctive polysomal pattern consisting of Another possibility is that this part of the den- short rows of granules attached to the endoplasmic drite is a growing or expanding tip of a growing or reticulum or small clusters suspended in the matrix regenerating nerve cell. If this possibility is ad- between cisterns. Although large polysomes are mitted for consideration, then dendrites become present, usually each polysome is composed of only more plastic and variable than they have been four to six ribosomes, considerably fewer than there thought to be. The presence of axonal terminals are in the polysomes of such neurons as the synapsing upon the surfaces of these dendrites does Purkinje cell. These results on sectioned material not argue against the possibility, for growth of the agree with the findings of Ekholm and Hyd6n (7), dendrites may be a method of establishing new who examined intact polysomes in fragments of synaptic contacts (see Footnote 6). Peculiar axonal isolated cells prepared by microdissection and terminals have also been seen in the lateral vestibu- negative staining. lar nucleus, as well as in many other parts of the Rich et al. (33) have clearly shown that the size central nervous system (11), and it has been sug- of the polysome is related to the complexity of the gested that these may represent stages in a cycle of protein molecule synthesized. For example, the reticulocyte, which synthesizes hemoglobin, has 6Through the courtesy of Dr. Kent Morest, of small polysomes composed of five to six ribosomes. this Department, we have recently had the privilege The messenger RNA that codes the polypeptide of examining dendrites in Golgi preparations of the chain of hemoglobin requires only 450 nucleotides. lateral vestibular and the lateral geniculate nuclei In contrast, in the HeLa cell infected with polio- of the cat. In young, growing kittens, the distal myelitis virus, where the synthesized protein is segments of dendrites display irregular weblike expansions and varicosities from which thin terminal very complex, the relevant messenger RNA processes extend. In 6-wk-old animals such formations requires 6000 nucleotides, and the polysomes in have not been seen as yet, but only the lateral genic- this instance are composed of 60 ribosomes. If, ulate nucleus has been examined at this time. then, the polysomal patterns of different cells can

176 THE JOURNAL OF CELL BIOLOGY VOLUME 36, 1968 be considered as reflecting the kinds and com- two cell types are linked together in the production plexities of the proteins synthesized in these cells, of energy, proteins, and bioelectric potentials. it is interesting to notice the variety of polysomal Although this theory is an elaboration of specula- arrays in different types of neurons. These patterns tions expressed earlier by Holmgren (13) and are not consistent with the size or volume of the Cajal (32), it has received a great deal of attention cell, as the pattern of the giant Deiters cell is similar from modern neurobiologists because of the elegant to that of cerebellar granule cells. The variety of microdissections and microchemical analyses that patterns suggests that neurons may produce dis- have been used to support it. tinctive proteins, specific to the cell type, or differ- The electron microscopic observations reported ent proportions of similar proteins of various com- in this paper have an important bearing on the plexities. applicability of cytochemical data to the support of this interesting speculation. Generally, the giant Relation Between Neurons and cell of Deiters is immediately surrounded by two Neuroglial Cells types of cellular component: (1) axon terminals

synapsing on its sufrace, and (2) delicate sheets of Downloaded from http://rupress.org/jcb/article-pdf/36/1/151/1068343/151.pdf by guest on 02 October 2021 In recent years, Hyden and his collaborators astrocytic cytoplasm. Oligodendrocytes rarely ap- (14, 15, 10) have carried out intensive neurochemi- proach the surface of the giant cell, and when they cal investigations on the lateral vestibular nucleus, do come close they are always separated from its particularly on the giant cells of Deiters and their surface by at least one thin astrocytic sheet. Thus, associated neuroglial cells. Hyden uses a delicate the true satellite of the giant cell is the astrocyte method of microdissection that permits him to and not the oligodendrocyte. This is in contrast isolate the giant neurons from their surrounding to the situation in the spinal cord, where both types neuroglial cells and thus to analyze each independ- of neuroglial cells can be in immediate contact ently. In the first studies (14, 15), a giant cell was with neuronal perikarya. The identification of the thought to be enclosed by a neuroglial envelope satellite is perhaps not critical for the success of the formed of about 35-40 oligodendrocytes, the pro- symbiotic theory, but the morphology of the satel- portion of astrocytes not exceeding 10%. More lite relationship is essential for interpreting the recently, Hamberger (10), using the same method, cytochemical data used to support it. The astro- found that the specimen considered as the peri- cytic neuroglial processes envelop not only the neuronal neuroglia of the Deiters cells contained perikaryon and the synaptic endings on its surface, only seven or eight neuroglial nuclei, but he con- but also a wide variety of structures including firmed the proportions of cell types given by Hyd6n myelinated nerve fibers, unmyelinated preterminal and Pigon (15), i.e., 90% oligodendrocytes and fibers, and dendrites of the same and other 10% astrocytes. Hyden and his collaborators have neurons. Although the cytochemical sample of the used these samples to analyze the content of oxida- perikaryon can be relatively clean, the neuroglial tive enzymes and the base composition of nucleic sample can only be heterogeneous. Thus, the re- acids in the neuroglial cells, compared with the sults of these cytochemical analyses are difficult tu giant neurons, under normal and experimental interpret, because the localization of enzynmatlc conditions, such as forced activity and learning. activities and other functions in such samples For example, they found that, in the normal ani- remains ambiguous. mal, cytochrome oxidase and succinoxidase ac- tivities are twice as intense in the satellite neuroglia as in an equal mass of Deiters neuron. Conversely, The investigations on which this report is based were the neuroglia contain only about 0.1 of the RNA supported, in part, by Public Health Service Re- search Grant No. B-3659 from the National Institute found in the neuron. Under certain types of chemi- of Neurological Diseases and Blindness, Bethesda, cal stimulation and forced activity, the base com- Maryland. Part of this work was carried out while position of RNA in the neurons and neuroglial Dr. Sotelo was the recipient of a Bourse de Recherche cells were reported to change in opposite direc- from the North Atlantic Treaty Organization. Dr. tions. On the basis of these and many other inge- Sotelo's present position is Attache de Recherche nious investigations, these authors have proposed a (C.N.R.S.) at the Laboratoire de Biologie Animale, general theory of a symbiotic relationship between Faculty des Sciences de Paris. neurons and satellite neuroglial cells in which the Received for publication 25 July 1967.

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C. SOTELO AND S. L. PALAY The Lateral Vestibular Nucleus. 1 179