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Neurofilaments and microtubules in anterior horn cells of the rat
T)SSUE 8- CELL 1969 "1 (3) 3 8 7 - 4 0 2 PuZ~hshedby Ohver & Boyd Ltd Edinburgh Printed m Great Brztam
RAYMOND
B WUERKER
and S A N F O R D
L
P A L A Y ~"
NEUROFILAMENTS AND MICROTUBULES IN A N T E R I O R HORN CELLS OF THE RAT ABSTRACT Dendrites ar{smg from t h e l a r g e r nerve cells)n the anterior horn conta)n fascmles of nearof)laments in add~tmn to the usual dendnt)c components From a comparison of neurof)laments and mmrotubules, and thmr respectwe subumts ~t )s concluded that d~rect mterconversmn between them hs improbable In transverse sectmn the wall of the neurofilament is composed of 4~6 mrcular denmttes about 30 A m diameter Short spokehke s)de arms p~oject from the mrcular densJhes )nto the surround)ng cottony matrix )n whmh the filaments are embedded
]ntroductton THE electron nucroscopic analysis of a region m the central nervous system depends primarily u p o n the r e c o g n m o n of profiles as belonging to ~denhfied n e u r o n s or, at the very least, to certain types of n e u r o n s In the neuropll, where the processes of nerve cells a n d n e u r o g h a l cells are intricately mtermingled, this r e c o g n m o n ~s difficult, because the small parts of the processes included within a thin section usually provide few dlst m c u v e clues to their ~dentlty The neurocytologist has, however, a n u m b e r of m e t h o d s for e s t a b h s h m g the c o n n e c n o n between a given profile a n d the cell, or type of cell, f r o m which ~t originates T h e sm~ptest a n d m o s t d u e c t m e t h o d ~s to trace the p~ocess to the cell of o n g m by m e a n s o f m o n t a g e s a n d serial sectmns, a difficult a n d laborious procedure t h a t ~s m o s t hkely to succeed m places where the process m question ~s relatively s h o r t and straight A n o t h e r m e t h o d ~s to int e r r u p t a b u n d l e of fibers surgically a n d t h e n to pick out m the electron m i c r o g l a p h s tile fibers a n d terminals t h a t are m a r k e d by the ensuing degeneratxon Th~s p r o c e d u r e can be . . . . . . . . . . . . . . . . . . . * Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115, U S A Received 27 January 1969 A
useful where a r a t h e r h o m o g e n e o u s a n d compact p a t h w a y ~s mvolved, a n d it is m any case restrmted to afferent axons Stdl a n o t h e r way ~s to locate in t h i n sections of a Golgi p~epazatlon a process previously identified in thick sections of t h e same p l e p a r a t l o n (Stell, 1965, AIksne et al, 1966) Thls lngemous m e t h o d Is very useful for selected areas in whmh the pattern of cell pl ocesses is already largely u n d e r s t o o d The easmst.method of all ~s to identify profiles o n the basis of their size, shape, p o s m o n , a n d internal structure, as given by hght microscopy Naturally, all of these m e t h o d s c a n be used together to solve a partJcular p r o b l e m But w~th each of theul the aim ~s the same, to locate a n d identify the parts o f specific cells, or types of cells, so that they can b e recogmzed m s t a n d a r d election m l c l o g l a p h s o f well preselved tissue A l t h o u g h one c a n n o t expect to find a specific fine structural p a t t e r n for every type o f n e r v e cell m the central n e r v o u s system, ~t ts n o t u n u s u a l to find t h a t m a particular region nerve cell processes can be r e a d d y ~ecogmzed by their size, shape, location, synaptlc relatmns, a n d internal s t r u c t u r e - - a l l c o m b i n i n g to produce a &stmctlve a n d rebible pattern Profiles of processes are more e a s i l y I d e n t i f i e d i n r e g i o n s where the n e r v e 387
W U E R K E R & PALAY
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motor neurons give off thick straight dendrites extending longitudinally among the other cells of the same column (Scheibel and Scheibel, 1966; Sterling and Kuypers, 1967). These longitudinally oriented dendrites tend to aggregate at the margin of the gray matter, where they can be examined in large numbers along with the perikarya from which they originate. By using sections cut across the axis of the spinal cord, it was possible to study the cells and dendrites of the same column in a single preparation. Thus it could be established that the dendrites of the large neurons in the anterior horn contain characteristic fascicles of neurofilaments in addition to the usual complement of microtubules. These arrays provided an opportunity to compare the fine structure of microtubules and neurofilaments in the same neuronal processes.
A v e r a ~ l e D i a m e t e r in ,u
Fig. 1, Histogram: Number of dendrites vs. their average diameter. The upper graph shows the average diameter of 39 dendritic profiles that contain scattered single and paired neurofilaments, The lower graph shows the diameters of 67 dendrites that contain numerous clusters of neurofilaments, These profiles were randomly selected from electron micrographs of transversely sectioned, longitudinal dendrites.
cells are set out in repetitive arrays, as in the cerebral and cerebellar cortices or in the retina. In regions that are ]less obviously ordered, the internal structure of processes can be a critical identifying feature, as the present study illustrates. In the anterior horn of the spinal cord, the large motor neurons are arranged in more or less distinct longitudinal columns, but their dendrites spread out in all directions, even reaching into the posterior horn (Valverde, 19,56). The analysis of the neuropil in the anterior horn would be considerably facilitated if it were found that the dendritic trees of the motor neurons had distinguishing internal morphological characteristics. We have approached this problem by taking advantage of the fact that many of the large
Methods
Cervical spinal cord segments were taken from 100-250 g albino rats (Charles River Breeding Laboratories, North Wilmington, Mass.). The animals were anesthetized by an intraperitoneal injection of chloral hydrate (0.35 mg per gram of body weight) and were perfused through the heart with a balanced salt solution (McEwen, 1956) followed immediately by fixative. The details of the perfusion procedure have been described previously (Palay et al., 1962). Two fixing solutions were used: either 3~0~ glutaraldehyde or a combination of 1/% paraformaldehyde and 1~/~ glutaraldehyde. The fixatives were made up in 0-12 M phosphate buffer (0.02 M NaH2PO4 and 0.10 M K2HPO4) containing 0.02 mM CaCI2. The glutaraldehyde was diluted from a stock 25% solution that had been passed through a column of Norit A. At the end of the perfusion the cervical spine and attached tissues were removed from the animal and left overnight in the same fixative. After about 18 hours the cord was dissected and sliced into blocks 1 m m thick with the faces of the blocks oriented parallel to or perpendicular to the longitudinal axis of the spinal cord. TISSUE Et CELL 1969 1 (3)
N E U R O F I L A M E N T S IN A N T E R I O R H O R N CELLS The blocks were rinsed in 0-12 M phosphate buffer containing 0.02 mM calcium chloride and 8%/, dextrose. They were then immersed for 2-4 hours in 2%; osmium tetroxide and 7~163 dextrose in the same buffer. After postfixation the blocks were washed in 2.4~.'~, NaC1 for 30 minutes, dehydrated in ascending concentrations of methanol, and embedded in Epon 812 (Shell). Sections 0.5 to 2/~ thick were cut with glass knives on a Huxley microtome, stained with 0-1,~ toluidine blue in 1~,;;borax, and studied with the light microscope. Light gold and silver sections were mounted on Formvarand carbon-coated grids, double stained with 4~,'s alcoholic uranyl acetate followed by 0-1 ?,o ~ lead citrate in 0.1 M sodium hydroxide, and examined in an RCA EMU-3G electron microscope. Golgi preparations were done according to the method of Morest and Morest (1966) or a modification of the Kopsch or Strong method (Addison, 1950). In the latter method, young rats weighing 120 g were perfused with a solution of 4~ paraformaldehyde in 3~;; potassium dichromate. The spinal cord was cut into blocks 3 mm thick and left to harden in the same solution for 3 days. The blocks were treated with 6~'.~potassium dichromate for 2 days followed by 0'75% silver nitrate for 3 days. They were then carried through increasing concentrations of ethanol into a 2 : 1 mixture of ethyl ether and ethanol, embedded in nitrocellulose, and finally sectioned at 100 t~. Observations Transverse sections of the anterior horn (Fig. 2) show that the large motor neurons in the cell columns are interspersed with fields of neuropil consisting largely of pale dendrites. Such fields are especially prominent in the lateral cell columns, and frequently they form the margin between the gray matter and the white. The clustering of the dendrites into islands of neuropil reflects the compact arrangement of the perikarya in tiers along the length of the spinal cord. Because the perikarya are staggered in this way, their TISSUE ~" CELL 1969 1 (3)
389
dendrites, extending longitudinally and ramifying within the confines of the cell column in which they originate, tend to run together in fasciculi. Thus a single transverse section across the anterior horn displays profiles of dendrites from different cells and at different levels in their course. In horizontal sections, cut in a plane parallel with the floor of the spinal canal (Fig. 3), these dendrites appear as long, straight bands of nearly uniform width, giving rise to only a few branches. A good example is the longitudinal dendrite shown in Fig. 5, which could be followed for 250 ~ in a single section. Many of its branches lie in the same plane as this section. Similarly, these longitudinally running dendrites could be followed for 325 to 1325 t~ in horizontally sectioned Golgi preparations. Here too, the majority of branches from these dendrites continue to run in a longitudinal direction while intermingling with similarly oriented dendrites of other cells in the same column. This is illustrated in Fig. 4, which is a montage of a Golgi-Kopsch preparation of three anterior horn cells and their longitudinally directed dendrites. As can b e seen in Figs. 2, 3, and 5, the fields of neuropil are interlaced with small and large myelinated fibers. These give rise to terminal unmyelinated branches that synapse with tile dendrites in a distinctive way. The dendrites are incrusted with terminals, both ~arge and small, crowding one against the other, with only thin astrocytic processes separating them. As the dendrites run parallel with one another and close together, most of the terminals are lodged in the interstices between them and many have synaptic faces attached to two or even three dendrites (see Fig. 7). The resultant synapses, spanning two or more dendrites, exemplify the most efficient method of ensuring the divergence of afferent impulses, an important integrative mechanism in the spinal cord (see Mendell and Henneman, 1968). This mode of termination is common in the islands of neuropil but not in the cell clusters, where the large perikarya are more widely Separated by myelinated and unmyelinated axons as well as by neuroglial cells.
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The fine structure o/" dendrites. Although dendrites in general contain most of the cytoplasmic organelles found in the perikaryon, they resemble the perikaryon closely only at their origins. The amounts of Nissl substance and Golgi complex diminish with distance fi'om the cell body. At the origin of a dendrite
the Nissl bodies and the Golgi apparatus are drawn into elongated masses. A Nissl body is often lodged at the more proximal branch points, like an island in a dividing stream. But farther along the Nissl substance becomes fragmented into very small masses--scattered elements of endoplasmic reticulum with a few
Light Micrographs Fig, 2. Transverse section of anterior horn. A group of ventral horn cells adjacent to the white matter (w) and surrounded by fields of densely packed dendrites (d). Stain : Toluidine blue. :~,770. Fig. 3. Horizontal section of anterior horn. Co}Is within a horizontal column are surrounded by bundles of longitudinally running dendrites (d). Stain: Toluidine blue. • 770. Fig. 4. Horizontal section of anterior horn, This montage is composed of micrographs taken at three different focal planes and shows longitudinally running dendrites (d~ and d~) from two different cells. Many of the branches from these dendrites also run longitudinally. Stain: Golgi-Kopsch. >: 310. Fig. 5. Horizontal section of anterior horn, This longitudinal dendrite (d) was traced for 250 F in the same 1 ~ section. Stain : Toluidine blue. > 720. Fig. 6. Transverse section of anterior horn, The Nissl bodies of the large cells have a "moth-eaten" appearance caused by lucent areas (arrows). In electron micrographs such as Fig. 12 these areas are filled by microtubules, neurofilaments, and smooth endoplasmic reticulum. Stain : Toluidine blue. ,- 3100.
Electron Micrographs Fig. 7. Synapse in transverse section of anterior horn. An ending makes synaptic contacts (*) with t w o different longitudinal dendrites (D~ and Do). Dendrite D1 contains numerous clusters of neurofilaments, which are indicated by arrows. ;< 33,000. Fig. 8, Dendritic cytoplasm within anterior horn. Microtubules (mt) are uniformly distributed throughout the cytoplasm. Their walls are composed of circular subunits with a dense core (rnta) or a lucent center (rot2). Neurofilaments appear tubular with a wall composed of circular densities (nf). Armlike spokes radiate from the neurofilaments (nf~) into the background matrix that surrounds the filaments2 x 280,000.
Electron Micrograph Fig. 9. Transverse section of two longitudinal dendrites in the anterior horn. The dendritic ground substance appears as a meshwork of threads (*) surrounding the microtubules (rot). This meshwork is interrupted by groups of neurofilaments (nf) that are embedded in a similar but more prominent matrix. The upper dendrite measures 1 F in diameter; the lower, 2 /~. x 120,000. TISSUE Et CELL 1969 1 (3)
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W U E R K E R & PALAY
The fine structure o/" dendrites. Although dendrites in general contain most of the cytoplasmic organelles found in the perikaryon, they resemble the perikaryon closely only at their origins. The amounts of Nissl substance and Golgi complex diminish with distance fi'om the cell body. At the origin of a dendrite
the Nissl bodies and the Golgi apparatus are drawn into elongated masses. A Nissl body is often lodged at the more proximal branch points, like an island in a dividing stream. But farther along the Nissl substance becomes fragmented into very small masses--scattered elements of endoplasmic reticulum with a few
Light Micrographs Fig, 2. Transverse section of anterior horn. A group of ventral horn cells adjacent to the white matter (w) and surrounded by fields of densely packed dendrites (d). Stain : Toluidine blue. :~,770. Fig. 3. Horizontal section of anterior horn. Co}Is within a horizontal column are surrounded by bundles of longitudinally running dendrites (d). Stain: Toluidine blue. • 770. Fig. 4. Horizontal section of anterior horn, This montage is composed of micrographs taken at three different focal planes and shows longitudinally running dendrites (d~ and d~) from two different cells. Many of the branches from these dendrites also run longitudinally. Stain: Golgi-Kopsch. >: 310. Fig. 5. Horizontal section of anterior horn, This longitudinal dendrite (d) was traced for 250 F in the same 1 ~ section. Stain : Toluidine blue. > 720. Fig. 6. Transverse section of anterior horn, The Nissl bodies of the large cells have a "moth-eaten" appearance caused by lucent areas (arrows). In electron micrographs such as Fig. 12 these areas are filled by microtubules, neurofilaments, and smooth endoplasmic reticulum. Stain : Toluidine blue. ,- 3100.
Electron Micrographs Fig. 7. Synapse in transverse section of anterior horn. An ending makes synaptic contacts (*) with t w o different longitudinal dendrites (D~ and Do). Dendrite D1 contains numerous clusters of neurofilaments, which are indicated by arrows. ;< 33,000. Fig. 8, Dendritic cytoplasm within anterior horn. Microtubules (mt) are uniformly distributed throughout the cytoplasm. Their walls are composed of circular subunits with a dense core (rnta) or a lucent center (rot2). Neurofilaments appear tubular with a wall composed of circular densities (nf). Armlike spokes radiate from the neurofilaments (nf~) into the background matrix that surrounds the filaments2 x 280,000.
Electron Micrograph Fig. 9. Transverse section of two longitudinal dendrites in the anterior horn. The dendritic ground substance appears as a meshwork of threads (*) surrounding the microtubules (rot). This meshwork is interrupted by groups of neurofilaments (nf) that are embedded in a similar but more prominent matrix. The upper dendrite measures 1 F in diameter; the lower, 2 /~. x 120,000. TISSUE Et CELL 1969 1 (3)
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ribosomes or merely some isolated rosettes of ribosomes--and finally it disappears altogether, Almost from the start most of the organelles assume a longitudinal orientation, with the result that in transverse sections of the anterior horn the intracellular structures in the dendrites are displayed in cross section, like the dendrites themselves (Figs. 7, 9, 10, 11). In dendrites, as in axons, there are four longitudinally oriented, anisometric organelles: the mitochondria, the agranular endoplasmic reticulum, the microtubules, and the neurofilaments. The mitochondria, at first short and plump, become more slender and elongated with increasing distance from the cell body; their cristae diminish in number and become more regularly oriented lengthwise. Their wide variation in size, even within the same dendrite, is remarkable (Figs. 7 and 10). They range from 0.1 to 0-5 ~r in diameter and from l to 10 in length. With increasing distance from the perikaryon the agranular endoplasmic reticulum becomes sparse and predominantly tubular with few branches, its diameter varying from 50 to 300 m~. The most conspicuous organdies in the dendrites are the microtubules. They run for long distances in nearly straight lines parallel to the long axis of the dendrite. Spaced across the dendrite at intervals of about 100 m#, they form in many places an array of ahnost crystalline regularity that is broken only by the intrusion of other organelles. This pattern is characteristic of dendrites, particularly of larger ones. In ahnost all preparations, the microtubules a r e surrounded by a cloud of fine threads and wisps that radiate out irregularly from them (Figs. 8-11). This delicate filamentous material forms a kind of web in which the micro-
tubules as well as the other organelles are suspended. From the evidence at hand it is impossible to ascertain whether this web represents preformed structure or an artefactitious precipitate of protein in the cytoplasmic matrix (see below). Unlike the microt u n e s in cilia or in the initial segment of the axon, the microtubules in the dendrites never appear bound together by arms or cross bars. Dendritic microtubules resemble those found in most other ceils (Gonatas and Robbins, 1965; Porter, 1965; Sandborn, 1966). They are roughly circular in cross section, about 270 ~ in diameter and they have a light core about 150 It in diarneter. The wall of the microtubule averages about 60 Zk in thickness and is composed of 11 to 14 circular subunits, which have either dense (m& in Fig. 8) or light (rag in Fig, 8) centers. It seems unlikely that the difference in the density of the subunits can be attributed merely to variable staining affinity, as suggested by Maser and Philpott (1966), for both light and dark subunits appear in the same profles. In transverse section, a few of the microtubules contain a central dot, about 50 A in diameter, which in appropriately oriental longitudinal sections proves to be a thin filament running in the core of the microtubule. Although neurofilaments are characteristic components of axons, they also occur in dendrites in small numbers scattered through the cytoplasm either singly or in pairs. They are, therefore, inconspicuous in most dendrites in nearly all parts of the central nervous system. In the ventral horn, however, only a minority of the dendrites follow this pattern. Most of the dendrites contain fascicles of neurofilaments, as many as 30 in a group, which run parallel to the axis of the dendrite
Electron M/crographs Fig. 10. Transversely sectioned dendrite in anterior horn. This dendrite is 2 /~ in diameter and contains numerous groups of neurofilaments as indicated by arrows. Note the meshwork surrounding the mierotubules (*). >: 55:000. Fig. 11. Transversely sectioned dendrite in anterior horn. This dendrite is 1 8 / z in diameter and contains only scattered single (nf) and paired (circled) neurofilaments. Note the meshwork surrounding the microtubules ( * ) . . : 55,000. TISSUE 8 CELL 1969 1 (3)
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ribosomes or merely some isolated rosettes of ribosomes--and finally it disappears altogether, Almost from the start most of the organelles assume a longitudinal orientation, with the result that in transverse sections of the anterior horn the intracellular structures in the dendrites are displayed in cross section, like the dendrites themselves (Figs. 7, 9, 10, 11). In dendrites, as in axons, there are four longitudinally oriented, anisometric organelles: the mitochondria, the agranular endoplasmic reticulum, the microtubules, and the neurofilaments. The mitochondria, at first short and plump, become more slender and elongated with increasing distance from the cell body; their cristae diminish in number and become more regularly oriented lengthwise. Their wide variation in size, even within the same dendrite, is remarkable (Figs. 7 and 10). They range from 0.1 to 0-5 ~r in diameter and from l to 10 in length. With increasing distance from the perikaryon the agranular endoplasmic reticulum becomes sparse and predominantly tubular with few branches, its diameter varying from 50 to 300 m~. The most conspicuous organdies in the dendrites are the microtubules. They run for long distances in nearly straight lines parallel to the long axis of the dendrite. Spaced across the dendrite at intervals of about 100 m#, they form in many places an array of ahnost crystalline regularity that is broken only by the intrusion of other organelles. This pattern is characteristic of dendrites, particularly of larger ones. In ahnost all preparations, the microtubules a r e surrounded by a cloud of fine threads and wisps that radiate out irregularly from them (Figs. 8-11). This delicate filamentous material forms a kind of web in which the micro-
tubules as well as the other organelles are suspended. From the evidence at hand it is impossible to ascertain whether this web represents preformed structure or an artefactitious precipitate of protein in the cytoplasmic matrix (see below). Unlike the microt u n e s in cilia or in the initial segment of the axon, the microtubules in the dendrites never appear bound together by arms or cross bars. Dendritic microtubules resemble those found in most other ceils (Gonatas and Robbins, 1965; Porter, 1965; Sandborn, 1966). They are roughly circular in cross section, about 270 ~ in diameter and they have a light core about 150 It in diarneter. The wall of the microtubule averages about 60 Zk in thickness and is composed of 11 to 14 circular subunits, which have either dense (m& in Fig. 8) or light (rag in Fig, 8) centers. It seems unlikely that the difference in the density of the subunits can be attributed merely to variable staining affinity, as suggested by Maser and Philpott (1966), for both light and dark subunits appear in the same profles. In transverse section, a few of the microtubules contain a central dot, about 50 A in diameter, which in appropriately oriental longitudinal sections proves to be a thin filament running in the core of the microtubule. Although neurofilaments are characteristic components of axons, they also occur in dendrites in small numbers scattered through the cytoplasm either singly or in pairs. They are, therefore, inconspicuous in most dendrites in nearly all parts of the central nervous system. In the ventral horn, however, only a minority of the dendrites follow this pattern. Most of the dendrites contain fascicles of neurofilaments, as many as 30 in a group, which run parallel to the axis of the dendrite
Electron M/crographs Fig. 10. Transversely sectioned dendrite in anterior horn. This dendrite is 2 /~ in diameter and contains numerous groups of neurofilaments as indicated by arrows. Note the meshwork surrounding the mierotubules (*). >: 55:000. Fig. 11. Transversely sectioned dendrite in anterior horn. This dendrite is 1 8 / z in diameter and contains only scattered single (nf) and paired (circled) neurofilaments. Note the meshwork surrounding the microtubules ( * ) . . : 55,000. TISSUE 8 CELL 1969 1 (3)
N E U R O F I L A M E N T S I N A N T E R I O R H O R N CELLS (Figs. 7-10). The fascicles are usually distributed at random within the dendrites, but in some they tend to be concentrated near the plasma membrane. In transverse section the neurofilaments appear as fine dots about 100/~ in diameter, spaced about 300 ~ from one another and embedded in a dense matrix (Fig. 7). At higher magnification the neurofilaments, like the microtubules, prove to have a circular cross section with a light core about 35 ~ in diameter. The wall of the profile appears to be composed of from 3 to 6 dense granules (Hf in Fig. 8) each about 30 ~ in diameter. As can be seen in Fig. 8, fine short rods of similar thickness radiate like spokes from the granules in the wall of the neurofilament 07f and nJ'~) into the surrounding matrix where they seem to be continuous with ill-defined wispy material in the background similar to that surrounding the microtubules. This material is very conspicuous where several neurofilaments occur close together as they do in the dendrites of anterior horn cells. In order to obtain some idea of the frequency of dendrites with such clustered neurofilaments, we took a sample of 106 transverse sections of dendrites from unselected micrographs of neuropil and divided the profiles into two groups according to their content of neurofilaments. Of these 106 dendritic profiles, 607.o contained one or more clusters of neurofilaments, and 40~ contained only the isolated single or paired neurofilaments (Fig. 11) that are typical of dendrites in other places, such as the cerebellar cortex and the frontal cerebral cortex. The diameters of the dendritic profiles with clustered filaments ranged from 0"7 to 11-3 #~. The dendrites with only scattered filaments or
pairs tended to be smaller, ranging from 0.2 ~ to 4"3 ~ in dialneter. As shown in Fig, 1, however, the two populations overlapped considerably in size, and this overlap indicates that although the largest profiles all contained clustered neurofilaments the presence or absence of these clusters is not simply correlated with the size of a dendrite. It must signify that dendrites with and without fascicles of neurofilaments originate from different kinds of nerve cells. We did not understand the significance of the clustered neurofilaments in the dendrites until we had studied a number of the perikarya in the anterior horn. The general fine structural features of this cell type have been described by Bodian (1964). Out of hundreds of cells examined in the electron microscope, we selected for systematic study eleven cells sectioned through their nucleoli. Montages were made of overlapping electron micrographs, and many of the cells were studied in more than one section. In these montages and serial sections, and in single micrographs of other cells, the neurofilaments and microtubules could be followed from the perikarya into the dendrites. In cell bodies 25 t~ or more in diameter (Fig. 12) clusters of parallel neurofilaments were found that resemble those described in the longitudinal dendrites. 3-hey have the same dimensions and substructure and are surrounded by a similar matrix. Thus, clusters of neurofilaments occur in large cells whence they continue into the longitudinal dendrites. In smaller cell bodies neurofilaments are dispersed singly and are hard to find (Fig. 13). The Nissl bodies of the large cells have certain special features. They are distinct massive blocks, separated from one another
Electron Micrograph Fig. 12. Nissl body in large anterior horn neuron, This clump of granular endoplasmic reticulum is punctuated by groups of neurofilaments (nf) and profiles of smooth endoplasmic reticulum (set). These structures together with microtubules give the Nissl body its "moth-eaten" appearance seen with the light microscope (see Fig, 6). In the cytoplasm surrounding the Nissl body there are groups of neurofilaments (nf) and microtubules (mt) that are both similar to those in dendrites, x 34,000. TISSUE 8- CELL 1969 1 (3)
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by extensive fields or 'roads" that are occupjed by heavy concentrations of microtubules, neurofilaments, mitochondria, and other organelles. ]t is in these spaces that the clustered neurofalaments lie (Fig. 12). Furthermore, in thin sections stained with toluidine blue and examined in the light microscope, these large Nissl bodies display small, clear, unstained holes (arrows in Fig. 6). This moth-eaten appearance is easily dismissed as indiscriminate swelling, as a fixation artefact. But on examination in the electron microscope, these holes turn out to be occupied by a few microtubules or by a cluster of neurofilaments like those in the dendrites (Fig. ~2). Accompanied by their special matrix, which excludes other organelles, the neurofilaments appear to penetrate the Nissl bodies in their course through the perikaryon. These fascicles of parallel neurofilaments must account for the familiar light microscope pattern of interlacing neurofibrillae, which usually do not leave room for massive Nissl bodies. Less commonly the holes are occupied by pieces of smooth endoplasmic reticuIum. In the smaller perikarya, ranging from 10 to 23 t~ in diameter, the Nissl substance is more finely divided and more uniformly distributed. It is pierced by single microtubules and occasionally by isolated neurofilaments (Fig. 13). Bundles of neurofilaments do not occur in these cells. From these observations it is reasonable to conclude tlnat the distinctive dendrites containing clustered neurofilaments are processes of the large motor neurons.
Discussion The neurofilaments in certain dendrites of the anterior horn are interesting from several
points of view. In the frst place dendrites rarely contain so many neurofilaments as these do. Dendrites in the cerebellar or cerebral cortices, the thalamus, and brain stem usually have so few neurofilaments that they are easily missed among the more characteristic and numerous microtubules. It is the axon that typically contains large numbers of neurofilaments. The inverse proportions of these two longitudinal organelles can be relied on to distinguish between axons and dendrites in many parts of the central nervous system. But this distinction is in fact only a generalization derived from a comparison of large myelinated axons with large dendrites. In the smallest axons, the most numerous axons in the central nervous system, neurofilaments are rarely found and microtubules are the principal organelles. In larger axons, although neurofilaments are usually numerous, so are the microtubules. Indeed, the proportions of these organelles vary so widely in different kinds of axons that the proportion in any axon can be considered as an identifying characteristic of the parent neuron rather than a correlate of axonal diameter (see, for example, the comparison of climbing fibers and basket cell axons by Larramendi et aL, 1967). In dendrites, however, neurofilaments are usually rare whatever the size and source of the dendrite, and the number of microtubules is correlated with diameter. As the dendrite ramifies and its diameter dwindles, microtubules (unlike mitochondria) decrease in number until they are infrequent in the terminal branchlets. The dendrites described in this paper are no exception to this general rule. Although they contain nurnerous neurofilaments, the microtubules remain preponderant. But the unusual number of neurofilaments in these dendrites suggests that they originate
Electron Micrograph Fig. 13. Cytoplasm of small anterior horn neuron. The Nissl substance is widely dispersed in ill-defined masses. The neurofilaments (nf) and microtubules (mt) are scarce and scattered t h r o u g h o u t the cytoplasm. >: 34,000.
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4O0 from a specific type of neuron. This suggestion was strengthened by the finding (Fig. 1) that all of the large dendrites in the anterior horn neuropil contain fascicles of neurofilaments, whereas the smaller dendrites may or may not have them. The tracing of the fascicles from the large neuronal perikarya into their dendrites and the absence of the fascicles from the smaller perikarya combine to demonstrate that the fascicles are characteristic features of the large cells and therefore identify the dendrites containing them as processes of the large cells. A second point of interest is the disposition of the filaments in the dendrites. They are arranged in fascicles that tend to lie in the peripheral cytoplasm. Within the fascicles, the filaments are set 300 A apart in a fine fibrillar matrix and they run roughly parallel with one another, twisting gradually. Thus, their disposition differs from that of neurofilaments in axons where they are usually in dividually and uniformly dispersed throughout the axoplasm, while the microtubules tend to lie peripherally. In this connection it may be recalled that according to Peters and Vaughn (1967) neurofilaments appear in axons of developing optic nerves first as fascicles and later as individuals dispersed throughout the axoplasm. The reciprocal relation between the neurofilaments and microtubules in terms of their position, number, and arrangement suggests a close genetic, chemical, and functional relation between them. There are, however, considerations that make it difficult to accept the conclusion that the two organelles are in some way interchangeable. From the morphological point of view the most important objection appears on comparison of their dimensions. Although both organelles are tubular in construction, microtubules have two or three times the diameter of neurofilaments and their walls are twice as thick. The wall of the microtubule is composed of 11-14 globular subunits about 60 A in diameter, whereas the wall of the neurofilaments is composed of 4-6 subunits only 30 A in diameter. A direct transformation of one into the other, as for example an
W U E R K E R & PALAY unravelling of the microtubule into neurofilaments, is therefore difficult to envisage. But it is plausible to think of the subunits disassembling under certain conditions into their unitary protein chains and then reassembling in a new conformation. So far this has not been demonstrated either experimentally or in the living animal. When microtubules have been depolymerized by temperature changes (Rodriguez Echandia and Piezzi, 1968) a fine, cottony fibrous material appears in their place and the neurofilaments remain unaffected. The same kind of result occurs when the fixation procedure has been inadequate. When the experimental conditions are reversed, microtubules form again with no change in the neurofilaments. Furthermore, although the amino acid compositions of microtubule protein and neurofilament protein are in many respects similar, they are not identical, and there are important chemical differences. For example, the microtubule protein binds guanine nucleotide, whereas the squid neurofilament protein contains no bound nucleotide. This subject is still under study by biochemists (see discussion in Schmitt and Samson, 1968). At present, however, the morphological evidence does not lend credence to a direct interchange or interconversion of the two organelles. A third aspect of interest is the function of the neurofilaments in the dendrites. In analogy with current speculations about microtubules, there is a strong tendency nowadays to attribute two important functions to neurofilaments: structural support and movement (see Schmitt and Samson, 1968). The long, straight (or gently twisting) fascicles of filaments can be seen as axial supports maintaining the severe anisometry of the dendrite as if it were threaded through with a set of rigid cables. This idea corresponds to an analogous interpretation of the microtubular arrays in the slender axopods of the heliozoan Acthlosphaerium (Tilney and Porter, 1967). But if microtubules are structural supports, then all dendrites are already well provided with them, and it is difficult to imagine the pertinence of an TISSUE 8- CELL 1969 1 (3)
NEUROFILAMENTS
IN A N T E R I O R H O R N CELLS
additional device for the same function. Besides, long dendrites of other types of large neurons, such as Purkinje cells, lack the fascicles of neurofilaments. The other suggestion, that the filaments are concerned with movement, is particularly appealing to morphologists since streaming and flowing movements of the neuronal cytoplasm have been well known for many years (see review by Barondes, 1967). Microscopic cinematography of living nerve cells and their processes (Pomerat et a[., 1967) reveals bidirectional streaming confined within channels in the perikaryon that correspond to the zones or 'roads' (Andres, 1961; Bunge et al., 1967) between Nissl bodies. In electron micrograpbs it can be seen that these 'roads' are largely occupied by neurofilaments. In addition, the present paper shows that fascicles of neurofilaments are threaded through the large Nissl bodies of anterior horn cells. This intimate association between the cytoplasmic proteinproducing machinery and the fascicles suggests that the neurofilaments are assembled in the channels within the Nissl bodies and from there are spun out into the rest of the cell and its processes. It is thus possible that the filaments in the Nissl bodies only indicate the site of production. But it is also plausible that they have a more select role in the diffusion of products from the perikaryon into the processes. The fascicles of neurofilaments might constitute a molecular motor for the transport of substances from the Nissl bodies where they are produced into the processes where they are
401
needed or consumed. The longitudinal arrangement of the filaments in fascicles might provide a mechanism for directional transport in contrast to simple non-directed diffusion. lit is in this connection that the side arms projecting from the neurofilaments attain the most interest. In muscle filaments the side arms or cross bridges contain ATPase which is believed to be involved in the translation of actin filaments upon myosin, so that muscle contraction results. Similarly, dynein, a protein with high ATPase activity, is found in the side arms of the outer microtubules in cilia and is believed to be involved in the motility of cilia. Finally, in squid neurofilament preparations a second protein has been found that contains phosphorylated serine and appears to be anisometric (P. F. Davison and F. C. Huneeus, unpublished results, quoted in Schmitt and Samson, 1968). This second protein might be homologous to muscle ATPase and dynein and might be the principal constituent of the side arms radiating from the neurofilaments. More detailed chemical and morphological studies are needed to characterize the side arms, as well as the filaments.
Acknowledgements This research was supported by U.S. Public Health Service grants NB03659 and NB 05591. Part of this work was carried out while Dr Wuerker was a Special Fellow of the National Institute of Neurological Diseases and Blindness (F10 NB 1468).
References ADDISON,W. H. F. 1950. Neurological technique. In McClung's Handbook of Microscopical Technique (3rd edition) (R. McC. Jones, editor). Paul B. Hoeber, Inc., New York. AL~:SNE,J. F., BLACKSTAD,R. W., WaL~Er~G, F. and WnTTE, L. E. JR. /966. Electron microscopy of a• degeneration: a valuable tool in experimental neuroanatomy. Ergebn. Anat. EntwGesch., 39, 1-32. ANDRES, K. H. 1961. Untersuchungen tiber den Feinbau yon Spinalganglien. Z. Zellforsch. mikrosk. Anat., 55, 1-48. BAROND~S,S. H. 1967. Axoplasmic transport. Neurosci. Res. Prog. BulL, 5, 307-419. BonIAN, D. 1964. An electron-microscopic study of the monkey spinal cord I. Fine structure of normal motor column. Bull. Johns Hopkins Hosp., 114, 13-119. TISSUE 8" CELL 1969 1 (3)
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BUNGE, M. B., BUNGE, R. P., PETERSON, E. R. and MURRAY, M. R. 1967. Light and electron microscope study of long-term organized cultures of rat dorsal root ganglia, d. Cell Biol., 32, 439-466. GONATAS, N. K. and RoumNS, E. 1965. The homology of spindle tubules and neurotubules in chick embryo retina. Protoplasma, 59, 377-391, LARRAMENDI, L. M. H. and VICTOR, T. 1967. Synapses on the Purkinje cell spines in the mouse. An electronmicroscopic study. Brain Res., 5, 15-30, MASER, M. D. and PI-IILPOT'F, C. W. 1966. The fine structure of marginal band microtubules. Anat. Ree., 154, 553-571. McEWEN, L. M. 1956, The effect on the isolated rabbit heart of vagal stimulation and its modification by cocaine, hexamethonium and ouabain. Y. Physiol., 131,678-689. MENDELL, L. M. and HENNEMAN,E. 1968. Terminals of single la fibres: distribution within a pool of 300 homonymous motor neurons. Xeienee, 160, 96-98. MoR~sx, D. K. and MoaEsr, R. R. 1966. Perfusion-fixation of the brain with chrome-osmium solutions for the rapid Golgi method. Am. d. Anat., 118, 811-832. PALAY, S. L., McGEE-Rt~ssELt,, S. M., GORDON, S. and GR1~_LO,M, A. 1962. Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide, d. Cell Biol., 12, 385410. PETERS, A. and VAUGnX, 31 E. 1967. Microtubules and filaments in axons and astrocytes of early postnatal rat optic nerves, d. Cell Biol., 32, 113-119. POMERAT, C. M., F~ENDELMAN,W. J., RAIBORN, C. W. and MASSeY, J. F. 1967. Dynamic activities of nervous tissue in vitro. In The Nem'on (H. Hyddn, editor), pp. 119-178. Elsevier, Amsterdam, PORTER, K. R, 1966. Cytoplasmic microtubules and their function. In Principles o/" Biomolecular Organization (G. E. W. Wolstenholme and M, O. O'Connor, editors), pp. 308-345. Little, Brown. Boston, Mass. Ror~R~c,uEz ECHANO~A,E. L. and PIEZZI, R. S. 1968. Microtubules in the nerve fibers of the toad Bq/b arenarum Hensel, Effect of low temperature on the sciatic nerve, a. Cell Biol., 39, 491-497. SANDBORY, E. B. 1966. Electron microscopy of the neuron membrane systems and filaments. Cart. J. Physiol. Pharmaco[., 44, 329-338, SChEmEr., M. E. and SCHF.mEL,A. B. 1966. Spinal motoneurons, interneurons, and Renshaw cells. A Golgi study. Archs ilal. Biol., 104, 328-353. SCHM[TT, F. O. and SAMSON,F. E. JR. 1968. Neuronal fibrous proteins. Neurosei. Res. Btdl., 6, 113-219. STELE, W. K. 1965. Correlation of retinal cytoarchitecture and ultrastructure in Golgi preparations. Anat. Rec., 153, 389-397. STERL~XG, P. and KUVPERS,H. G. J. M, 1967. Anatomical organization of the brachia[ spinal cord of the cat. 11. The motoneuron p]exus. Brain Res., 4, 16-32. T1tNE% L. G. and PORTER, K. R. 1967. Studies on the microtubules in heliozoa, 11. The effect of low temperature on these structures in the formation and maintenance of the axopodia. J. Cell Biol., 34. 327- 343. VALVEROE,F. 1966. The pyramidial tract in rodents. A study of its relations with the posterior column nuclei, dorsofateral reticular formation of the medulla oblongata, and cervical spinal cord (Golgi and electron microscopic observations). Z. ZellJbrsch. mikrosk. Anat., 71,297-363.
TISSUE & CELL 1,969 1 (3)
RAYMOND
B WUERKER
and S A N F O R D
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NEUROFILAMENTS AND MICROTUBULES IN A N T E R I O R HORN CELLS OF THE RAT ABSTRACT Dendrites ar{smg from t h e l a r g e r nerve cells)n the anterior horn conta)n fascmles of nearof)laments in add~tmn to the usual dendnt)c components From a comparison of neurof)laments and mmrotubules, and thmr respectwe subumts ~t )s concluded that d~rect mterconversmn between them hs improbable In transverse sectmn the wall of the neurofilament is composed of 4~6 mrcular denmttes about 30 A m diameter Short spokehke s)de arms p~oject from the mrcular densJhes )nto the surround)ng cottony matrix )n whmh the filaments are embedded
]ntroductton THE electron nucroscopic analysis of a region m the central nervous system depends primarily u p o n the r e c o g n m o n of profiles as belonging to ~denhfied n e u r o n s or, at the very least, to certain types of n e u r o n s In the neuropll, where the processes of nerve cells a n d n e u r o g h a l cells are intricately mtermingled, this r e c o g n m o n ~s difficult, because the small parts of the processes included within a thin section usually provide few dlst m c u v e clues to their ~dentlty The neurocytologist has, however, a n u m b e r of m e t h o d s for e s t a b h s h m g the c o n n e c n o n between a given profile a n d the cell, or type of cell, f r o m which ~t originates T h e sm~ptest a n d m o s t d u e c t m e t h o d ~s to trace the p~ocess to the cell of o n g m by m e a n s o f m o n t a g e s a n d serial sectmns, a difficult a n d laborious procedure t h a t ~s m o s t hkely to succeed m places where the process m question ~s relatively s h o r t and straight A n o t h e r m e t h o d ~s to int e r r u p t a b u n d l e of fibers surgically a n d t h e n to pick out m the electron m i c r o g l a p h s tile fibers a n d terminals t h a t are m a r k e d by the ensuing degeneratxon Th~s p r o c e d u r e can be . . . . . . . . . . . . . . . . . . . * Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115, U S A Received 27 January 1969 A
useful where a r a t h e r h o m o g e n e o u s a n d compact p a t h w a y ~s mvolved, a n d it is m any case restrmted to afferent axons Stdl a n o t h e r way ~s to locate in t h i n sections of a Golgi p~epazatlon a process previously identified in thick sections of t h e same p l e p a r a t l o n (Stell, 1965, AIksne et al, 1966) Thls lngemous m e t h o d Is very useful for selected areas in whmh the pattern of cell pl ocesses is already largely u n d e r s t o o d The easmst.method of all ~s to identify profiles o n the basis of their size, shape, p o s m o n , a n d internal structure, as given by hght microscopy Naturally, all of these m e t h o d s c a n be used together to solve a partJcular p r o b l e m But w~th each of theul the aim ~s the same, to locate a n d identify the parts o f specific cells, or types of cells, so that they can b e recogmzed m s t a n d a r d election m l c l o g l a p h s o f well preselved tissue A l t h o u g h one c a n n o t expect to find a specific fine structural p a t t e r n for every type o f n e r v e cell m the central n e r v o u s system, ~t ts n o t u n u s u a l to find t h a t m a particular region nerve cell processes can be r e a d d y ~ecogmzed by their size, shape, location, synaptlc relatmns, a n d internal s t r u c t u r e - - a l l c o m b i n i n g to produce a &stmctlve a n d rebible pattern Profiles of processes are more e a s i l y I d e n t i f i e d i n r e g i o n s where the n e r v e 387
W U E R K E R & PALAY
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motor neurons give off thick straight dendrites extending longitudinally among the other cells of the same column (Scheibel and Scheibel, 1966; Sterling and Kuypers, 1967). These longitudinally oriented dendrites tend to aggregate at the margin of the gray matter, where they can be examined in large numbers along with the perikarya from which they originate. By using sections cut across the axis of the spinal cord, it was possible to study the cells and dendrites of the same column in a single preparation. Thus it could be established that the dendrites of the large neurons in the anterior horn contain characteristic fascicles of neurofilaments in addition to the usual complement of microtubules. These arrays provided an opportunity to compare the fine structure of microtubules and neurofilaments in the same neuronal processes.
A v e r a ~ l e D i a m e t e r in ,u
Fig. 1, Histogram: Number of dendrites vs. their average diameter. The upper graph shows the average diameter of 39 dendritic profiles that contain scattered single and paired neurofilaments, The lower graph shows the diameters of 67 dendrites that contain numerous clusters of neurofilaments, These profiles were randomly selected from electron micrographs of transversely sectioned, longitudinal dendrites.
cells are set out in repetitive arrays, as in the cerebral and cerebellar cortices or in the retina. In regions that are ]less obviously ordered, the internal structure of processes can be a critical identifying feature, as the present study illustrates. In the anterior horn of the spinal cord, the large motor neurons are arranged in more or less distinct longitudinal columns, but their dendrites spread out in all directions, even reaching into the posterior horn (Valverde, 19,56). The analysis of the neuropil in the anterior horn would be considerably facilitated if it were found that the dendritic trees of the motor neurons had distinguishing internal morphological characteristics. We have approached this problem by taking advantage of the fact that many of the large
Methods
Cervical spinal cord segments were taken from 100-250 g albino rats (Charles River Breeding Laboratories, North Wilmington, Mass.). The animals were anesthetized by an intraperitoneal injection of chloral hydrate (0.35 mg per gram of body weight) and were perfused through the heart with a balanced salt solution (McEwen, 1956) followed immediately by fixative. The details of the perfusion procedure have been described previously (Palay et al., 1962). Two fixing solutions were used: either 3~0~ glutaraldehyde or a combination of 1/% paraformaldehyde and 1~/~ glutaraldehyde. The fixatives were made up in 0-12 M phosphate buffer (0.02 M NaH2PO4 and 0.10 M K2HPO4) containing 0.02 mM CaCI2. The glutaraldehyde was diluted from a stock 25% solution that had been passed through a column of Norit A. At the end of the perfusion the cervical spine and attached tissues were removed from the animal and left overnight in the same fixative. After about 18 hours the cord was dissected and sliced into blocks 1 m m thick with the faces of the blocks oriented parallel to or perpendicular to the longitudinal axis of the spinal cord. TISSUE Et CELL 1969 1 (3)
N E U R O F I L A M E N T S IN A N T E R I O R H O R N CELLS The blocks were rinsed in 0-12 M phosphate buffer containing 0.02 mM calcium chloride and 8%/, dextrose. They were then immersed for 2-4 hours in 2%; osmium tetroxide and 7~163 dextrose in the same buffer. After postfixation the blocks were washed in 2.4~.'~, NaC1 for 30 minutes, dehydrated in ascending concentrations of methanol, and embedded in Epon 812 (Shell). Sections 0.5 to 2/~ thick were cut with glass knives on a Huxley microtome, stained with 0-1,~ toluidine blue in 1~,;;borax, and studied with the light microscope. Light gold and silver sections were mounted on Formvarand carbon-coated grids, double stained with 4~,'s alcoholic uranyl acetate followed by 0-1 ?,o ~ lead citrate in 0.1 M sodium hydroxide, and examined in an RCA EMU-3G electron microscope. Golgi preparations were done according to the method of Morest and Morest (1966) or a modification of the Kopsch or Strong method (Addison, 1950). In the latter method, young rats weighing 120 g were perfused with a solution of 4~ paraformaldehyde in 3~;; potassium dichromate. The spinal cord was cut into blocks 3 mm thick and left to harden in the same solution for 3 days. The blocks were treated with 6~'.~potassium dichromate for 2 days followed by 0'75% silver nitrate for 3 days. They were then carried through increasing concentrations of ethanol into a 2 : 1 mixture of ethyl ether and ethanol, embedded in nitrocellulose, and finally sectioned at 100 t~. Observations Transverse sections of the anterior horn (Fig. 2) show that the large motor neurons in the cell columns are interspersed with fields of neuropil consisting largely of pale dendrites. Such fields are especially prominent in the lateral cell columns, and frequently they form the margin between the gray matter and the white. The clustering of the dendrites into islands of neuropil reflects the compact arrangement of the perikarya in tiers along the length of the spinal cord. Because the perikarya are staggered in this way, their TISSUE ~" CELL 1969 1 (3)
389
dendrites, extending longitudinally and ramifying within the confines of the cell column in which they originate, tend to run together in fasciculi. Thus a single transverse section across the anterior horn displays profiles of dendrites from different cells and at different levels in their course. In horizontal sections, cut in a plane parallel with the floor of the spinal canal (Fig. 3), these dendrites appear as long, straight bands of nearly uniform width, giving rise to only a few branches. A good example is the longitudinal dendrite shown in Fig. 5, which could be followed for 250 ~ in a single section. Many of its branches lie in the same plane as this section. Similarly, these longitudinally running dendrites could be followed for 325 to 1325 t~ in horizontally sectioned Golgi preparations. Here too, the majority of branches from these dendrites continue to run in a longitudinal direction while intermingling with similarly oriented dendrites of other cells in the same column. This is illustrated in Fig. 4, which is a montage of a Golgi-Kopsch preparation of three anterior horn cells and their longitudinally directed dendrites. As can b e seen in Figs. 2, 3, and 5, the fields of neuropil are interlaced with small and large myelinated fibers. These give rise to terminal unmyelinated branches that synapse with tile dendrites in a distinctive way. The dendrites are incrusted with terminals, both ~arge and small, crowding one against the other, with only thin astrocytic processes separating them. As the dendrites run parallel with one another and close together, most of the terminals are lodged in the interstices between them and many have synaptic faces attached to two or even three dendrites (see Fig. 7). The resultant synapses, spanning two or more dendrites, exemplify the most efficient method of ensuring the divergence of afferent impulses, an important integrative mechanism in the spinal cord (see Mendell and Henneman, 1968). This mode of termination is common in the islands of neuropil but not in the cell clusters, where the large perikarya are more widely Separated by myelinated and unmyelinated axons as well as by neuroglial cells.
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The fine structure o/" dendrites. Although dendrites in general contain most of the cytoplasmic organelles found in the perikaryon, they resemble the perikaryon closely only at their origins. The amounts of Nissl substance and Golgi complex diminish with distance fi'om the cell body. At the origin of a dendrite
the Nissl bodies and the Golgi apparatus are drawn into elongated masses. A Nissl body is often lodged at the more proximal branch points, like an island in a dividing stream. But farther along the Nissl substance becomes fragmented into very small masses--scattered elements of endoplasmic reticulum with a few
Light Micrographs Fig, 2. Transverse section of anterior horn. A group of ventral horn cells adjacent to the white matter (w) and surrounded by fields of densely packed dendrites (d). Stain : Toluidine blue. :~,770. Fig. 3. Horizontal section of anterior horn. Co}Is within a horizontal column are surrounded by bundles of longitudinally running dendrites (d). Stain: Toluidine blue. • 770. Fig. 4. Horizontal section of anterior horn, This montage is composed of micrographs taken at three different focal planes and shows longitudinally running dendrites (d~ and d~) from two different cells. Many of the branches from these dendrites also run longitudinally. Stain: Golgi-Kopsch. >: 310. Fig. 5. Horizontal section of anterior horn, This longitudinal dendrite (d) was traced for 250 F in the same 1 ~ section. Stain : Toluidine blue. > 720. Fig. 6. Transverse section of anterior horn, The Nissl bodies of the large cells have a "moth-eaten" appearance caused by lucent areas (arrows). In electron micrographs such as Fig. 12 these areas are filled by microtubules, neurofilaments, and smooth endoplasmic reticulum. Stain : Toluidine blue. ,- 3100.
Electron Micrographs Fig. 7. Synapse in transverse section of anterior horn. An ending makes synaptic contacts (*) with t w o different longitudinal dendrites (D~ and Do). Dendrite D1 contains numerous clusters of neurofilaments, which are indicated by arrows. ;< 33,000. Fig. 8, Dendritic cytoplasm within anterior horn. Microtubules (mt) are uniformly distributed throughout the cytoplasm. Their walls are composed of circular subunits with a dense core (rnta) or a lucent center (rot2). Neurofilaments appear tubular with a wall composed of circular densities (nf). Armlike spokes radiate from the neurofilaments (nf~) into the background matrix that surrounds the filaments2 x 280,000.
Electron Micrograph Fig. 9. Transverse section of two longitudinal dendrites in the anterior horn. The dendritic ground substance appears as a meshwork of threads (*) surrounding the microtubules (rot). This meshwork is interrupted by groups of neurofilaments (nf) that are embedded in a similar but more prominent matrix. The upper dendrite measures 1 F in diameter; the lower, 2 /~. x 120,000. TISSUE Et CELL 1969 1 (3)
390
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The fine structure o/" dendrites. Although dendrites in general contain most of the cytoplasmic organelles found in the perikaryon, they resemble the perikaryon closely only at their origins. The amounts of Nissl substance and Golgi complex diminish with distance fi'om the cell body. At the origin of a dendrite
the Nissl bodies and the Golgi apparatus are drawn into elongated masses. A Nissl body is often lodged at the more proximal branch points, like an island in a dividing stream. But farther along the Nissl substance becomes fragmented into very small masses--scattered elements of endoplasmic reticulum with a few
Light Micrographs Fig, 2. Transverse section of anterior horn. A group of ventral horn cells adjacent to the white matter (w) and surrounded by fields of densely packed dendrites (d). Stain : Toluidine blue. :~,770. Fig. 3. Horizontal section of anterior horn. Co}Is within a horizontal column are surrounded by bundles of longitudinally running dendrites (d). Stain: Toluidine blue. • 770. Fig. 4. Horizontal section of anterior horn, This montage is composed of micrographs taken at three different focal planes and shows longitudinally running dendrites (d~ and d~) from two different cells. Many of the branches from these dendrites also run longitudinally. Stain: Golgi-Kopsch. >: 310. Fig. 5. Horizontal section of anterior horn, This longitudinal dendrite (d) was traced for 250 F in the same 1 ~ section. Stain : Toluidine blue. > 720. Fig. 6. Transverse section of anterior horn, The Nissl bodies of the large cells have a "moth-eaten" appearance caused by lucent areas (arrows). In electron micrographs such as Fig. 12 these areas are filled by microtubules, neurofilaments, and smooth endoplasmic reticulum. Stain : Toluidine blue. ,- 3100.
Electron Micrographs Fig. 7. Synapse in transverse section of anterior horn. An ending makes synaptic contacts (*) with t w o different longitudinal dendrites (D~ and Do). Dendrite D1 contains numerous clusters of neurofilaments, which are indicated by arrows. ;< 33,000. Fig. 8, Dendritic cytoplasm within anterior horn. Microtubules (mt) are uniformly distributed throughout the cytoplasm. Their walls are composed of circular subunits with a dense core (rnta) or a lucent center (rot2). Neurofilaments appear tubular with a wall composed of circular densities (nf). Armlike spokes radiate from the neurofilaments (nf~) into the background matrix that surrounds the filaments2 x 280,000.
Electron Micrograph Fig. 9. Transverse section of two longitudinal dendrites in the anterior horn. The dendritic ground substance appears as a meshwork of threads (*) surrounding the microtubules (rot). This meshwork is interrupted by groups of neurofilaments (nf) that are embedded in a similar but more prominent matrix. The upper dendrite measures 1 F in diameter; the lower, 2 /~. x 120,000. TISSUE Et CELL 1969 1 (3)
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ribosomes or merely some isolated rosettes of ribosomes--and finally it disappears altogether, Almost from the start most of the organelles assume a longitudinal orientation, with the result that in transverse sections of the anterior horn the intracellular structures in the dendrites are displayed in cross section, like the dendrites themselves (Figs. 7, 9, 10, 11). In dendrites, as in axons, there are four longitudinally oriented, anisometric organelles: the mitochondria, the agranular endoplasmic reticulum, the microtubules, and the neurofilaments. The mitochondria, at first short and plump, become more slender and elongated with increasing distance from the cell body; their cristae diminish in number and become more regularly oriented lengthwise. Their wide variation in size, even within the same dendrite, is remarkable (Figs. 7 and 10). They range from 0.1 to 0-5 ~r in diameter and from l to 10 in length. With increasing distance from the perikaryon the agranular endoplasmic reticulum becomes sparse and predominantly tubular with few branches, its diameter varying from 50 to 300 m~. The most conspicuous organdies in the dendrites are the microtubules. They run for long distances in nearly straight lines parallel to the long axis of the dendrite. Spaced across the dendrite at intervals of about 100 m#, they form in many places an array of ahnost crystalline regularity that is broken only by the intrusion of other organelles. This pattern is characteristic of dendrites, particularly of larger ones. In ahnost all preparations, the microtubules a r e surrounded by a cloud of fine threads and wisps that radiate out irregularly from them (Figs. 8-11). This delicate filamentous material forms a kind of web in which the micro-
tubules as well as the other organelles are suspended. From the evidence at hand it is impossible to ascertain whether this web represents preformed structure or an artefactitious precipitate of protein in the cytoplasmic matrix (see below). Unlike the microt u n e s in cilia or in the initial segment of the axon, the microtubules in the dendrites never appear bound together by arms or cross bars. Dendritic microtubules resemble those found in most other ceils (Gonatas and Robbins, 1965; Porter, 1965; Sandborn, 1966). They are roughly circular in cross section, about 270 ~ in diameter and they have a light core about 150 It in diarneter. The wall of the microtubule averages about 60 Zk in thickness and is composed of 11 to 14 circular subunits, which have either dense (m& in Fig. 8) or light (rag in Fig, 8) centers. It seems unlikely that the difference in the density of the subunits can be attributed merely to variable staining affinity, as suggested by Maser and Philpott (1966), for both light and dark subunits appear in the same profles. In transverse section, a few of the microtubules contain a central dot, about 50 A in diameter, which in appropriately oriental longitudinal sections proves to be a thin filament running in the core of the microtubule. Although neurofilaments are characteristic components of axons, they also occur in dendrites in small numbers scattered through the cytoplasm either singly or in pairs. They are, therefore, inconspicuous in most dendrites in nearly all parts of the central nervous system. In the ventral horn, however, only a minority of the dendrites follow this pattern. Most of the dendrites contain fascicles of neurofilaments, as many as 30 in a group, which run parallel to the axis of the dendrite
Electron M/crographs Fig. 10. Transversely sectioned dendrite in anterior horn. This dendrite is 2 /~ in diameter and contains numerous groups of neurofilaments as indicated by arrows. Note the meshwork surrounding the mierotubules (*). >: 55:000. Fig. 11. Transversely sectioned dendrite in anterior horn. This dendrite is 1 8 / z in diameter and contains only scattered single (nf) and paired (circled) neurofilaments. Note the meshwork surrounding the microtubules ( * ) . . : 55,000. TISSUE 8 CELL 1969 1 (3)
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ribosomes or merely some isolated rosettes of ribosomes--and finally it disappears altogether, Almost from the start most of the organelles assume a longitudinal orientation, with the result that in transverse sections of the anterior horn the intracellular structures in the dendrites are displayed in cross section, like the dendrites themselves (Figs. 7, 9, 10, 11). In dendrites, as in axons, there are four longitudinally oriented, anisometric organelles: the mitochondria, the agranular endoplasmic reticulum, the microtubules, and the neurofilaments. The mitochondria, at first short and plump, become more slender and elongated with increasing distance from the cell body; their cristae diminish in number and become more regularly oriented lengthwise. Their wide variation in size, even within the same dendrite, is remarkable (Figs. 7 and 10). They range from 0.1 to 0-5 ~r in diameter and from l to 10 in length. With increasing distance from the perikaryon the agranular endoplasmic reticulum becomes sparse and predominantly tubular with few branches, its diameter varying from 50 to 300 m~. The most conspicuous organdies in the dendrites are the microtubules. They run for long distances in nearly straight lines parallel to the long axis of the dendrite. Spaced across the dendrite at intervals of about 100 m#, they form in many places an array of ahnost crystalline regularity that is broken only by the intrusion of other organelles. This pattern is characteristic of dendrites, particularly of larger ones. In ahnost all preparations, the microtubules a r e surrounded by a cloud of fine threads and wisps that radiate out irregularly from them (Figs. 8-11). This delicate filamentous material forms a kind of web in which the micro-
tubules as well as the other organelles are suspended. From the evidence at hand it is impossible to ascertain whether this web represents preformed structure or an artefactitious precipitate of protein in the cytoplasmic matrix (see below). Unlike the microt u n e s in cilia or in the initial segment of the axon, the microtubules in the dendrites never appear bound together by arms or cross bars. Dendritic microtubules resemble those found in most other ceils (Gonatas and Robbins, 1965; Porter, 1965; Sandborn, 1966). They are roughly circular in cross section, about 270 ~ in diameter and they have a light core about 150 It in diarneter. The wall of the microtubule averages about 60 Zk in thickness and is composed of 11 to 14 circular subunits, which have either dense (m& in Fig. 8) or light (rag in Fig, 8) centers. It seems unlikely that the difference in the density of the subunits can be attributed merely to variable staining affinity, as suggested by Maser and Philpott (1966), for both light and dark subunits appear in the same profles. In transverse section, a few of the microtubules contain a central dot, about 50 A in diameter, which in appropriately oriental longitudinal sections proves to be a thin filament running in the core of the microtubule. Although neurofilaments are characteristic components of axons, they also occur in dendrites in small numbers scattered through the cytoplasm either singly or in pairs. They are, therefore, inconspicuous in most dendrites in nearly all parts of the central nervous system. In the ventral horn, however, only a minority of the dendrites follow this pattern. Most of the dendrites contain fascicles of neurofilaments, as many as 30 in a group, which run parallel to the axis of the dendrite
Electron M/crographs Fig. 10. Transversely sectioned dendrite in anterior horn. This dendrite is 2 /~ in diameter and contains numerous groups of neurofilaments as indicated by arrows. Note the meshwork surrounding the mierotubules (*). >: 55:000. Fig. 11. Transversely sectioned dendrite in anterior horn. This dendrite is 1 8 / z in diameter and contains only scattered single (nf) and paired (circled) neurofilaments. Note the meshwork surrounding the microtubules ( * ) . . : 55,000. TISSUE 8 CELL 1969 1 (3)
N E U R O F I L A M E N T S I N A N T E R I O R H O R N CELLS (Figs. 7-10). The fascicles are usually distributed at random within the dendrites, but in some they tend to be concentrated near the plasma membrane. In transverse section the neurofilaments appear as fine dots about 100/~ in diameter, spaced about 300 ~ from one another and embedded in a dense matrix (Fig. 7). At higher magnification the neurofilaments, like the microtubules, prove to have a circular cross section with a light core about 35 ~ in diameter. The wall of the profile appears to be composed of from 3 to 6 dense granules (Hf in Fig. 8) each about 30 ~ in diameter. As can be seen in Fig. 8, fine short rods of similar thickness radiate like spokes from the granules in the wall of the neurofilament 07f and nJ'~) into the surrounding matrix where they seem to be continuous with ill-defined wispy material in the background similar to that surrounding the microtubules. This material is very conspicuous where several neurofilaments occur close together as they do in the dendrites of anterior horn cells. In order to obtain some idea of the frequency of dendrites with such clustered neurofilaments, we took a sample of 106 transverse sections of dendrites from unselected micrographs of neuropil and divided the profiles into two groups according to their content of neurofilaments. Of these 106 dendritic profiles, 607.o contained one or more clusters of neurofilaments, and 40~ contained only the isolated single or paired neurofilaments (Fig. 11) that are typical of dendrites in other places, such as the cerebellar cortex and the frontal cerebral cortex. The diameters of the dendritic profiles with clustered filaments ranged from 0"7 to 11-3 #~. The dendrites with only scattered filaments or
pairs tended to be smaller, ranging from 0.2 ~ to 4"3 ~ in dialneter. As shown in Fig, 1, however, the two populations overlapped considerably in size, and this overlap indicates that although the largest profiles all contained clustered neurofilaments the presence or absence of these clusters is not simply correlated with the size of a dendrite. It must signify that dendrites with and without fascicles of neurofilaments originate from different kinds of nerve cells. We did not understand the significance of the clustered neurofilaments in the dendrites until we had studied a number of the perikarya in the anterior horn. The general fine structural features of this cell type have been described by Bodian (1964). Out of hundreds of cells examined in the electron microscope, we selected for systematic study eleven cells sectioned through their nucleoli. Montages were made of overlapping electron micrographs, and many of the cells were studied in more than one section. In these montages and serial sections, and in single micrographs of other cells, the neurofilaments and microtubules could be followed from the perikarya into the dendrites. In cell bodies 25 t~ or more in diameter (Fig. 12) clusters of parallel neurofilaments were found that resemble those described in the longitudinal dendrites. 3-hey have the same dimensions and substructure and are surrounded by a similar matrix. Thus, clusters of neurofilaments occur in large cells whence they continue into the longitudinal dendrites. In smaller cell bodies neurofilaments are dispersed singly and are hard to find (Fig. 13). The Nissl bodies of the large cells have certain special features. They are distinct massive blocks, separated from one another
Electron Micrograph Fig. 12. Nissl body in large anterior horn neuron, This clump of granular endoplasmic reticulum is punctuated by groups of neurofilaments (nf) and profiles of smooth endoplasmic reticulum (set). These structures together with microtubules give the Nissl body its "moth-eaten" appearance seen with the light microscope (see Fig, 6). In the cytoplasm surrounding the Nissl body there are groups of neurofilaments (nf) and microtubules (mt) that are both similar to those in dendrites, x 34,000. TISSUE 8- CELL 1969 1 (3)
397
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WUERKER & PALAY
by extensive fields or 'roads" that are occupjed by heavy concentrations of microtubules, neurofilaments, mitochondria, and other organelles. ]t is in these spaces that the clustered neurofalaments lie (Fig. 12). Furthermore, in thin sections stained with toluidine blue and examined in the light microscope, these large Nissl bodies display small, clear, unstained holes (arrows in Fig. 6). This moth-eaten appearance is easily dismissed as indiscriminate swelling, as a fixation artefact. But on examination in the electron microscope, these holes turn out to be occupied by a few microtubules or by a cluster of neurofilaments like those in the dendrites (Fig. ~2). Accompanied by their special matrix, which excludes other organelles, the neurofilaments appear to penetrate the Nissl bodies in their course through the perikaryon. These fascicles of parallel neurofilaments must account for the familiar light microscope pattern of interlacing neurofibrillae, which usually do not leave room for massive Nissl bodies. Less commonly the holes are occupied by pieces of smooth endoplasmic reticuIum. In the smaller perikarya, ranging from 10 to 23 t~ in diameter, the Nissl substance is more finely divided and more uniformly distributed. It is pierced by single microtubules and occasionally by isolated neurofilaments (Fig. 13). Bundles of neurofilaments do not occur in these cells. From these observations it is reasonable to conclude tlnat the distinctive dendrites containing clustered neurofilaments are processes of the large motor neurons.
Discussion The neurofilaments in certain dendrites of the anterior horn are interesting from several
points of view. In the frst place dendrites rarely contain so many neurofilaments as these do. Dendrites in the cerebellar or cerebral cortices, the thalamus, and brain stem usually have so few neurofilaments that they are easily missed among the more characteristic and numerous microtubules. It is the axon that typically contains large numbers of neurofilaments. The inverse proportions of these two longitudinal organelles can be relied on to distinguish between axons and dendrites in many parts of the central nervous system. But this distinction is in fact only a generalization derived from a comparison of large myelinated axons with large dendrites. In the smallest axons, the most numerous axons in the central nervous system, neurofilaments are rarely found and microtubules are the principal organelles. In larger axons, although neurofilaments are usually numerous, so are the microtubules. Indeed, the proportions of these organelles vary so widely in different kinds of axons that the proportion in any axon can be considered as an identifying characteristic of the parent neuron rather than a correlate of axonal diameter (see, for example, the comparison of climbing fibers and basket cell axons by Larramendi et aL, 1967). In dendrites, however, neurofilaments are usually rare whatever the size and source of the dendrite, and the number of microtubules is correlated with diameter. As the dendrite ramifies and its diameter dwindles, microtubules (unlike mitochondria) decrease in number until they are infrequent in the terminal branchlets. The dendrites described in this paper are no exception to this general rule. Although they contain nurnerous neurofilaments, the microtubules remain preponderant. But the unusual number of neurofilaments in these dendrites suggests that they originate
Electron Micrograph Fig. 13. Cytoplasm of small anterior horn neuron. The Nissl substance is widely dispersed in ill-defined masses. The neurofilaments (nf) and microtubules (mt) are scarce and scattered t h r o u g h o u t the cytoplasm. >: 34,000.
TISSUE 8- CELL 1969 1 (3)
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4O0 from a specific type of neuron. This suggestion was strengthened by the finding (Fig. 1) that all of the large dendrites in the anterior horn neuropil contain fascicles of neurofilaments, whereas the smaller dendrites may or may not have them. The tracing of the fascicles from the large neuronal perikarya into their dendrites and the absence of the fascicles from the smaller perikarya combine to demonstrate that the fascicles are characteristic features of the large cells and therefore identify the dendrites containing them as processes of the large cells. A second point of interest is the disposition of the filaments in the dendrites. They are arranged in fascicles that tend to lie in the peripheral cytoplasm. Within the fascicles, the filaments are set 300 A apart in a fine fibrillar matrix and they run roughly parallel with one another, twisting gradually. Thus, their disposition differs from that of neurofilaments in axons where they are usually in dividually and uniformly dispersed throughout the axoplasm, while the microtubules tend to lie peripherally. In this connection it may be recalled that according to Peters and Vaughn (1967) neurofilaments appear in axons of developing optic nerves first as fascicles and later as individuals dispersed throughout the axoplasm. The reciprocal relation between the neurofilaments and microtubules in terms of their position, number, and arrangement suggests a close genetic, chemical, and functional relation between them. There are, however, considerations that make it difficult to accept the conclusion that the two organelles are in some way interchangeable. From the morphological point of view the most important objection appears on comparison of their dimensions. Although both organelles are tubular in construction, microtubules have two or three times the diameter of neurofilaments and their walls are twice as thick. The wall of the microtubule is composed of 11-14 globular subunits about 60 A in diameter, whereas the wall of the neurofilaments is composed of 4-6 subunits only 30 A in diameter. A direct transformation of one into the other, as for example an
W U E R K E R & PALAY unravelling of the microtubule into neurofilaments, is therefore difficult to envisage. But it is plausible to think of the subunits disassembling under certain conditions into their unitary protein chains and then reassembling in a new conformation. So far this has not been demonstrated either experimentally or in the living animal. When microtubules have been depolymerized by temperature changes (Rodriguez Echandia and Piezzi, 1968) a fine, cottony fibrous material appears in their place and the neurofilaments remain unaffected. The same kind of result occurs when the fixation procedure has been inadequate. When the experimental conditions are reversed, microtubules form again with no change in the neurofilaments. Furthermore, although the amino acid compositions of microtubule protein and neurofilament protein are in many respects similar, they are not identical, and there are important chemical differences. For example, the microtubule protein binds guanine nucleotide, whereas the squid neurofilament protein contains no bound nucleotide. This subject is still under study by biochemists (see discussion in Schmitt and Samson, 1968). At present, however, the morphological evidence does not lend credence to a direct interchange or interconversion of the two organelles. A third aspect of interest is the function of the neurofilaments in the dendrites. In analogy with current speculations about microtubules, there is a strong tendency nowadays to attribute two important functions to neurofilaments: structural support and movement (see Schmitt and Samson, 1968). The long, straight (or gently twisting) fascicles of filaments can be seen as axial supports maintaining the severe anisometry of the dendrite as if it were threaded through with a set of rigid cables. This idea corresponds to an analogous interpretation of the microtubular arrays in the slender axopods of the heliozoan Acthlosphaerium (Tilney and Porter, 1967). But if microtubules are structural supports, then all dendrites are already well provided with them, and it is difficult to imagine the pertinence of an TISSUE 8- CELL 1969 1 (3)
NEUROFILAMENTS
IN A N T E R I O R H O R N CELLS
additional device for the same function. Besides, long dendrites of other types of large neurons, such as Purkinje cells, lack the fascicles of neurofilaments. The other suggestion, that the filaments are concerned with movement, is particularly appealing to morphologists since streaming and flowing movements of the neuronal cytoplasm have been well known for many years (see review by Barondes, 1967). Microscopic cinematography of living nerve cells and their processes (Pomerat et a[., 1967) reveals bidirectional streaming confined within channels in the perikaryon that correspond to the zones or 'roads' (Andres, 1961; Bunge et al., 1967) between Nissl bodies. In electron micrograpbs it can be seen that these 'roads' are largely occupied by neurofilaments. In addition, the present paper shows that fascicles of neurofilaments are threaded through the large Nissl bodies of anterior horn cells. This intimate association between the cytoplasmic proteinproducing machinery and the fascicles suggests that the neurofilaments are assembled in the channels within the Nissl bodies and from there are spun out into the rest of the cell and its processes. It is thus possible that the filaments in the Nissl bodies only indicate the site of production. But it is also plausible that they have a more select role in the diffusion of products from the perikaryon into the processes. The fascicles of neurofilaments might constitute a molecular motor for the transport of substances from the Nissl bodies where they are produced into the processes where they are
401
needed or consumed. The longitudinal arrangement of the filaments in fascicles might provide a mechanism for directional transport in contrast to simple non-directed diffusion. lit is in this connection that the side arms projecting from the neurofilaments attain the most interest. In muscle filaments the side arms or cross bridges contain ATPase which is believed to be involved in the translation of actin filaments upon myosin, so that muscle contraction results. Similarly, dynein, a protein with high ATPase activity, is found in the side arms of the outer microtubules in cilia and is believed to be involved in the motility of cilia. Finally, in squid neurofilament preparations a second protein has been found that contains phosphorylated serine and appears to be anisometric (P. F. Davison and F. C. Huneeus, unpublished results, quoted in Schmitt and Samson, 1968). This second protein might be homologous to muscle ATPase and dynein and might be the principal constituent of the side arms radiating from the neurofilaments. More detailed chemical and morphological studies are needed to characterize the side arms, as well as the filaments.
Acknowledgements This research was supported by U.S. Public Health Service grants NB03659 and NB 05591. Part of this work was carried out while Dr Wuerker was a Special Fellow of the National Institute of Neurological Diseases and Blindness (F10 NB 1468).
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& PALAY
BUNGE, M. B., BUNGE, R. P., PETERSON, E. R. and MURRAY, M. R. 1967. Light and electron microscope study of long-term organized cultures of rat dorsal root ganglia, d. Cell Biol., 32, 439-466. GONATAS, N. K. and RoumNS, E. 1965. The homology of spindle tubules and neurotubules in chick embryo retina. Protoplasma, 59, 377-391, LARRAMENDI, L. M. H. and VICTOR, T. 1967. Synapses on the Purkinje cell spines in the mouse. An electronmicroscopic study. Brain Res., 5, 15-30, MASER, M. D. and PI-IILPOT'F, C. W. 1966. The fine structure of marginal band microtubules. Anat. Ree., 154, 553-571. McEWEN, L. M. 1956, The effect on the isolated rabbit heart of vagal stimulation and its modification by cocaine, hexamethonium and ouabain. Y. Physiol., 131,678-689. MENDELL, L. M. and HENNEMAN,E. 1968. Terminals of single la fibres: distribution within a pool of 300 homonymous motor neurons. Xeienee, 160, 96-98. MoR~sx, D. K. and MoaEsr, R. R. 1966. Perfusion-fixation of the brain with chrome-osmium solutions for the rapid Golgi method. Am. d. Anat., 118, 811-832. PALAY, S. L., McGEE-Rt~ssELt,, S. M., GORDON, S. and GR1~_LO,M, A. 1962. Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide, d. Cell Biol., 12, 385410. PETERS, A. and VAUGnX, 31 E. 1967. Microtubules and filaments in axons and astrocytes of early postnatal rat optic nerves, d. Cell Biol., 32, 113-119. POMERAT, C. M., F~ENDELMAN,W. J., RAIBORN, C. W. and MASSeY, J. F. 1967. Dynamic activities of nervous tissue in vitro. In The Nem'on (H. Hyddn, editor), pp. 119-178. Elsevier, Amsterdam, PORTER, K. R, 1966. Cytoplasmic microtubules and their function. In Principles o/" Biomolecular Organization (G. E. W. Wolstenholme and M, O. O'Connor, editors), pp. 308-345. Little, Brown. Boston, Mass. Ror~R~c,uEz ECHANO~A,E. L. and PIEZZI, R. S. 1968. Microtubules in the nerve fibers of the toad Bq/b arenarum Hensel, Effect of low temperature on the sciatic nerve, a. Cell Biol., 39, 491-497. SANDBORY, E. B. 1966. Electron microscopy of the neuron membrane systems and filaments. Cart. J. Physiol. Pharmaco[., 44, 329-338, SChEmEr., M. E. and SCHF.mEL,A. B. 1966. Spinal motoneurons, interneurons, and Renshaw cells. A Golgi study. Archs ilal. Biol., 104, 328-353. SCHM[TT, F. O. and SAMSON,F. E. JR. 1968. Neuronal fibrous proteins. Neurosei. Res. Btdl., 6, 113-219. STELE, W. K. 1965. Correlation of retinal cytoarchitecture and ultrastructure in Golgi preparations. Anat. Rec., 153, 389-397. STERL~XG, P. and KUVPERS,H. G. J. M, 1967. Anatomical organization of the brachia[ spinal cord of the cat. 11. The motoneuron p]exus. Brain Res., 4, 16-32. T1tNE% L. G. and PORTER, K. R. 1967. Studies on the microtubules in heliozoa, 11. The effect of low temperature on these structures in the formation and maintenance of the axopodia. J. Cell Biol., 34. 327- 343. VALVEROE,F. 1966. The pyramidial tract in rodents. A study of its relations with the posterior column nuclei, dorsofateral reticular formation of the medulla oblongata, and cervical spinal cord (Golgi and electron microscopic observations). Z. ZellJbrsch. mikrosk. Anat., 71,297-363.
TISSUE & CELL 1,969 1 (3)