A Light and Electron Microscope Study of Long

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A LIGHT AND ELECTRON MICROSCOPE

STUDY OF LONG-TERM ORGANIZED CULTURES

OF RAT DORSAL ROOT GANGLIA

MARY BARTLETT BUNGE, RICHARD P. BUNGE, EDITH R. PETERSON, and MARGARET R. MURRAY

From the Departments of Anatomy and Surgery, Columbia University College of Physicians and Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 Surgeons, New York

ABSTRACT Dorsal root ganglia from fetal rats were explanted on collagen-coated coverslips and carried in Maximow double-coverslip assemblies for periods up to 3 months. These cultured ganglia were studied in the living state, in stained whole mounts, and in sections after OsO4 fixation and Epon embedment. From the central cluster of nerve cell bodies, neurites emerge to form a rich network of fascicles which often reach the edge of the carrying coverslip. The neurons resemble their in vivo counterparts in nuclear and cytoplasmic content and organization; e.g., they appear as "light" or "dark" cells, depending on the amount of cytoplasmic neurofilaments. Satellite cells form a complete investment around the neuronal soma and are themselves everywhere covered by basement membrane. The neuron-satellite cell boundary is complicated by spinelike processes arising from the neuronal soma. Neuron size, myelinated fiber diameter, and internode length in the cultures do not reach the larger of the values known for ganglion and peripheral nerve in situ (30). Unmyelinated and myelinated nerve fibers and associated Schwann cells and endoneurial and perineurial components are organized into typical fascicles. The relationship of the Schwann cell and its single myelinated fiber or numerous unmyelinated fibers and the properties of myelin, such as lamellar spacing, mesaxons, Schmidt-Lanterman clefts, nodes of Ranvier, and protuberances, mimic the in vivo pattern. It is concluded that cultivation of fetal rat dorsal root ganglia by this technique fosters maturation and long-term maintenance of all the elements that comprise this cellular community in vivo (except vascular components) and, furthermore, allows these various components to relate faithfully to one another to produce an organotypic model of sensory ganglion tissue.

INTRODUCTION A series of technical advances (reviewed in 1) has one of the most useful cytological models. The made possible the maturation and long-term explant becomes thin enough for direct high-power maintenance of many parts of the nervous system light microscopic observation of neurons in the in culture: sympathetic ganglia (2-4), spinal (5) living state. Highly organized outgrowth areas and cranial ganglia (6, 7), spinal cord (8), cerebel- offer a similar opportunity for direct observation lum (9-11), brain stem (12), and cerebrum (13), of unmyelinated and myelinated neurites and their among others. Of these various types of nervous supporting elements. Such cultures have proven tissue preparations, the sensory ganglion provides useful for (a) direct observation of cytoplasmic

439 movements presumably involved in transport (Holmes' silver), or connective tissue elements within the neuron and axon (14); (b) studies of (Foot-Bielschowsky). the relation of the Schwann cell to the myelin For electron microscopy, representative ganglia sheath during myelinogenesis (15, 16), and (c) ob- were selected at intervals after 4 wk in vitro. (Neu- ronal maturation and widespread myelination re- servations on the pattern of myelin or neuron re- quire 6 8 wk in culture.) They were rinsed free of sponse to various insults (reviewed in 1 plus recent feeding medium with BSS, fixed in Veronal acetate- references 6, 17, 18). buffered OsO4, dehydrated in ethanol, embedded In order to establish the suitability of this in in Epon, sectioned for light or electron microscopy, vitro system for experimental electron microscopic and examined in a Zeiss light mieroscope or an studies, it was necessary to extend the investigation RCA EMU 3G electron microscope. The details of cytological fidelity to the uhrastructural level. of handling this type of culture for electron micros- Preliminary observations had indicated that cul- copy are given in reference 26.1 22 of the cultures tured dorsal root ganglia were highly promising so prepared were studied electron microscopically for such studies (19, 20). Rat rather than chick (after 24, 32, 33, 37, 39, 42, 56, 72, 77, or 79 days in vitro). No indication of infection by bacteria or ganglion cultures were chosen for electron micro- higher microorganisms was encountered in the scopic study because formation of myelin sheaths electron micrographs. Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 and organization of nerve fibers into fascicles are The cultures were thus analyzed: (a) in the living more extensive (see below). There are now avail- state in the Maximow assembly, (b) in the whole ble a number of papers describing the fine struc- mount after fixation and staining on the original ture of adult rat spinal ganglion in situ (reviewed coverslip, (c) in tolnidine blue-stained (28) 1 or in 21 and 22), and it is the purpose of this com- 2 /z-sections of plastic-embedded cultures ("semi- munication to demonstrate fine structural similar- thin sections"), and/or (d) in lead citrate~tained ities between these normal adult ganglia and their (29) thin sections viewed in the electron microscope. long-term cultured counterparts. This is the first In c and d, realignment of the tissue blocks as described in reference 26 allowed examination of the of a group of ultrastructural studies of cultured rat culture in desired planes of section. dorsal root ganglia. The reports immediately following (23, 24) present a detailed description of OBSERVATIONS AND COMMENTS the effects of in vitro X-irradiation on the mature cultures. In explanting the dorsal root ganglion, the central and peripheral branches of the dorsal root are cut MATERIALS AND METHODS near the edge of the ganglion. In culture the neurons remain in a central, more or less compact Cervical or lumbar (occasionally thoracic) dorsal mass while the nerve fibers grow out for a distance root ganglia were removed from fetal rats after 16~-19 days in utero. Ganglia taken from the older of 5-10 mm, thus reaching areas near the edge of fetuses were preferred for electron microscopic the coverslip. These neurites may sprout from the study because they retain, even in long-term cul- cut ends of the original root or they may radiate tures, a number of layers of neurons in the explaot from all parts of the explant. The outgrowing area. One or two ganglia were placed on a No. 1 round (22 mm in diameter) collagen-coated (25) 1 The method involves enlbedding the culture coverslip and maintained in a Maximow double- without its removal from the coverslip. The con- coverslip assembly. The cultures were fed at ex- sistently good fixation of this cultured tissue may be plantation and twice a week thereafter with one due, at least in part, to the lack of mechanical drop of a complex feeding mixture, after washing in handling, an idea likewise entertained by Webster balanced salt solution (BSS). This feed consisted and Collins (81) for in situ ganglion. The problem of one part human placental serum, one part 9-day of removing the carrying coverslip from the culture chick embryo extract, one part bovine serum ultra- after embedding directly on glass was solved by filtrate, and one part BSS. The mixture was supple- Robbins mad Gonatas (27) by initially coating the mented with glucose to give a final concentration glass surface with a heavy coat of carbon. Their of 600 mg % and with penicillin to give a concentra- technique works well in the present culture system, tion of about 200 units per ml. Light microscopic but the carbon coat must be limited to the central observations were made directly on the living culture region of the earrying eoverslip to give the collagen (up to 600 X magnification) or on the whole mount coat an adequate foothold on the coverslip periphery. after fixation and staining for myelin (Sudan black Our present experience indicates that cultures de- B), Nissl substance (toluidine blue), nerve fibers velop normally on coverslips so prepared.

440 TnE JOURNAL OF CELL BIOLOGY • VOLUME8~, 1967 nerve processes may traverse a zone near the ex- discrete Nissl bodies scattered throughout the plant in considerable disarray and then become cytoplasm or, in some of the larger neurons, con- organized into bundles more peripherally (Fig. 1). centrated more peripherally. Each bundle contains both myelinated and un- The general organization and placement of myelinated fibers and their accompanying neurons is best seen in semi-thin sections (Figs. 2, 4). Schwann cells. The neuronal mass is encapsulated In cross-section, the neurons can be seen grouped by connective tissue (Fig. 2), and fiber bundles are or scattered in a number of layers among fibers associated with perineurial sheaths (Fig. 10). Each and Schwann cells, all of these structures encased of these areas will be described separately with within a sheath of flattened connective tissue. The correlated light and electron microscopic observa- smaller neurons tend to be grouped together more tions under the headings: neuronal somas, satellite closely and, possibly as a consequence, tend to be cells, and nerve fiber bundles. more angular in shape than the larger neurons (as in reference 21). The neurons are not flattened Neuronal Somas in the plane of the coverslip. Cytoplasmic organiza- In the living culture (Fig. 3), neuronal somas are tion varies as illustrated in Fig. 4. Neuron 1 (nl) is small and contains fairly dense and homogeneous seen in a scattered monolayer or, more often, in a Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 number of layers. The neurons do not migrate. cytoplasm. Neuron 2 (n~), on the other hand, is The somas are round, elliptical, or angular in larger with lighter and less homogeneous cyto- shape and measure 11.5-50 # in diameter (30), plasm in which one can discern small light patches, as compared to < 18-75 /z for adult rat lumbar particularly in the perinuclear region. These light ganglia neurons in vivo (21). The vesicular nucleus spots correspond to the areas containing neuroffla- occupies about one-half the neuron diameter in ments as seen in the electron microscope. In neuron the smaller neurons, about one-third in the larger. 3, the differing granularity of the perinuclear and It is near the cell center but rarely appears exactly peripheral zones is more marked. Neuron 4, a central; it is almost always eccentric in the smallest binucleate cell, demonstrates that a lighter axon neurons. The rounded nuclear profile rarely shows hillock area may sometimes be found in these indentations. Most cultures contain a few blnucle- preparations and that the exiting axon is straight, ate neurons which are the largest in size. The nu- as is often the case. Coiling of the axon into a cleolus, one to three per nucleus, is clearly resolved glomerulus has not been observed in any of the against the lighter nucleoplasm and occupies light microscopic preparations thus far. Fig. 4 about one-fourth the diameter of the nucleus. The also illustrates the frequent position of the nucleo- cytoplasm displays a fine granularity which is lus flattened against the nuclear membrane, as more concentrated in the perinuclear area. The Andres (21) noted in situ. Bourne and Tewari quality of this granularity is considered to be an (31) have ascribed functional significance to this index of the general health of the neuron--when it configuration in dorsal root ganglion neurons in becomes more or less extensive or coarser or be- vivo. comes differently distributed, the health of the In the electron microscope, the spheroidal or ellip- neuron is in question. A culture which contains tical profile of the nucleus may occasionally dis- 3-5 % neurons with altered cytoplasmic granularity play irregular shallow undulations or indentations is not considered abnormal. (Fig. 5); only rarely are deep indentations or folds In whole mount preparations stained with Sudan visible. The nucleus is usually situated in a para- black, the normal fine cytoplasmic granularity is central rather than central position as Andres (21) particularly well seen. The moderately dense gran- found in rat spinal ganglia in situ. Chromatin is ules are generally dispersed throughout the cyto- finely dispersed with only occasional loci of clump- plasm, but in some large neurons they are more ing, generally not near the nuclear envelope. Often concentrated around the nucleus. Occasionally, one or two nucleoli are visible. The nucleolus is one sees, scattered through the cytoplasm, more made up of the usual components, i.e., dense gran- dense 1-2 # droplets thought to be neutral fat. ules and less well-defined units of smaller diameter. Clear axon hillock regions are seldom found and The latter are disposed as dense aggregates or, less the path of the emerging axon is difficult to follow frequently, as light patches, and are presumed to in these preparations. In formalin-fixed whole be the filaments observed by others. Nucleolar com- mounts stained for Nissl substance, neurons show ponents are organized into a tightly woven nu-

BUNGE, BUN'OE, PETERSON, AND MuanAY Rat Dorsal Root Ganglion Cultures 441 Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021

FIG~n~ 1 This is a survey photomicrograph of a dorsal root ganglion culture. Extending from the central dense area, which contains the neuronal cell bodies, are nerve fibers gathered into fascicles (arrows) of varying thickness. Some fibers pass through a zone (best shown at *) where they lose their faseicular arrangement and occur singly. About one-half the length of the peripherally directed fascicles is shown; these fascicles terminate near the edge of the carrying eoverslip. Adventitial cell nuclei create the background pattern. Whole mount fixed with Os04 and stained with Sudan black. >( £0. cleolonema, Protuberances from nucleoli and nu- may be dotted with ribosomes or may display cleolar satellites noted in vivo (21) were not ob- vesicle-like or cisternal outpocketings. served. Often skirting the nueleolus, and in other In thin sections, mitochondria appear round, ellip- areas of the nucleus as well, are loosely clus- soidal or rodlike, with cristae mitochondriales tered, irregularly shaped, highly dense particles oriented in different directions but usually in a ~,400 A in diameter. Comparable particles have plane perpendicular to their length. The mito- been noted in rat spinal ganglia in vivo (32). chondrial matrix is moderately dense and contains The nuclear envelope is studded with pores. particles of much greater density (Fig. 7). These When the envelope has been sectioned tangentially mitochondria are typical for neurons (34). Oc- (Fig. 7), the pores are seen to be 680-1050 or 300- casionally, irregularly contoured mitochondria 500 A in outside or inside diameter, respectively, display a matrix of diminished density with cristae and may display a central density. Dimensions mitochondriales situated mainly peripherally. reported by others studying mammalian spinal They may occur near normal-appearing mito- ganglion neurons in vivo are: 850 A (outside di- chondria. Similar mitochondria were found by ameter, 33), 280-360 A (inside diameter, 34) and Cerv6s-Navarro (22) in rat spinal ganglia in vivo 1000-1100 A or 400-500 A (outside or inside di- though he noted that these two forms of mito- ameter, 21). The inner membrane of the envelope chondria did not occur in the same neuron. appears thicker, denser and smoother than the A Golgi area appears as a typical straight or ruffled outer membrane, as Palay and Palade (34) curved complex of closely packed cisternae and and Andres (21) pointed out. The outer membrane vacuoles surrounded by sprays of small vesicles

442 THE JounNAb OF CELL BIoLoGY • VOLUME3~, 1967 (Fig. 7). In a thin section, a number of such com- ular bodies. Others of varying shape are uniformly plexes are scattered around the nucleus (Fig. 6); dense or m@ exhibit aggregates of highly dense their placement in a complex reticular network particles and/or stacked linear densities (Fig. 7). around the nucleus as shown by the light micros- They lack the peripherally located vacuoles of copist (35) can be easily envisioned. In rare cases, contrasting density which characterize the lipo- a cisterna appears partially "coated." Coated fuscin found by other investigators (21, 22, 37, 38) vesicles are frequently encountered in the vicinity in mammalian spinal ganglion neurons; this is of these Golgi complexes (or near the plasma- undoubtedly due to the young age of the cultured lemma). Dense-cored vesicles may be present in tissue. Lipid droplets, encountered occasionally in Golgi areas and, in rare instances, appear coated. the perinuclear or peripheral cytoplasm, have a Also associated with the Golgi complex are the dense, wavy profile and an empty-appearing in- multivesicular bodies first described in neurons by terior. Centrioles and cilia are present very rarely. Palay and Palade (34). At one extreme, the multi- Nissl substance, or granular endoplasmic reticu- vesicular body may be large, irregularly shaped, lum (34), occurs throughout small somas or prin- and seemingly empty but for a vesicle or two. At cipally in the periphery of large perikarya. In the other, it appears as a smaller sphere filled with small cell bodies, the Nissl substance appears uni- Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 dark vesicles or discs and patches of dense sub- formly distributed (Fig. 5) whereas in the larger stance arranged in a dense matrix (comparable to somas it may appear as small discrete bodies, as "dark" and "light" multivesicular bodies, ref. 36). large aggregates (ns, Fig. 6), or more rarely as a All types may display vesicle-like outpocketings thick, dense, nearly uninterrupted ring as Andres and a plaque (rarely two) of fuzzy or radially (21) noted in rat dorsal root ganglia in situ. striated dense substance on the outside of a flat- The discrete Nissl bodies are noted in those somas tened portion of the limiting membrane (Fig. 7). containing prominent neurofilaments. Nissl sub- Such configurations have been described primarily stance often extends to the plasmalemma. The in central nervous tissue (reviewed in 26) but also cisternae and vacuoles of endoplasmic reticulum in toad spinal ganglion (36). Multivesicular bodies are usually only partially covered by ribosomes; appear most frequently in association with the most ribosomes, in the form of rosettes, lie free in the Golgi complex or in the vicinity of the plasma- intervening cytoplasmic matrix. The cisternae usu- lemma. (Those near the somal surface more often ally display little apparent orientation when viewed seem to be of the irregularly shaped, empty-ap- in a plane either perpendicular or parallel to the pearing type.) coverslip on which they are grown, but they occa- Dense bodies (lysosomes?), seen by many electron sionally may be situated in the parallel arrays illus- microscopists studying neurons, are scattered, trated for rat dorsal root ganglia in vivo (22, 34, 80). usually sparsely, throughout the perinuclear cyto- When the cisternal membrane has been grazed plasm. Some of the smaller ones contain barely during sectioning (Fig. 7) the ribosomes are in typ- discernible vesicles, reminiscent of the multivesic- ical single or double rows which may be straight,

FmvaE ~ A particularly thick explant region is illustrated in this figure. This stained semi-thin section was cut in a plane perpendicular to the glass coverslip. The explant region, composed of neurons and numerous fiber bundles, is surrounded by a sheath of highly flattened cells (arrows). The bottom of the culture is at the lower edge of the figure. Beneath the culture the collagen film coating the coverslip appears as a clear zone (*).)< 360.

BUNGE, BUNGE, PETERSON, AND MURRAY R~t Dorsal Root Ganglion Cultures 443 Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021

FIOURE 3 This photomicrograph shows the appearance of living neurons. Nucleoli are prominent in the light nucleoplasm; the cytoplasmic granularity illustrated here is typical for healthy neurons. Most of the cells shown have assumed an angular outline. Related satellite cells (arrows) and a myelin sheath (my) may be seen. Bright-field microscopy with 40 X fluorite oil lens with 1 mm working distance. X 1,050. curved, e- or c-shaped, or looped, or form a circle conclusions drawn from a study of in situ rat tissue as in situ (34). (39). Subsurface cisternae are present as small vacuoles, Typical neurofilaments are present, predominantly as larger vacuoles or cisternae partially covered in the wide perinuclear zone with the mitochondria, by ribosomes (Fig. 7), or as ribosome-lacklng, Golgi apparatus, and dense bodies (Fig. 7). Their highly flattened cisternae which follow closely~ the diameter generally falls into the 70-100 A size ruffled contour of the plasmalemma. Very rarely range indicated by others (21, 34). The smallest a cisterna is seen to veer away from the subsurface neurons contain only a few which are hardly position into the cytoplasm where it widens and noticeable among the other cytoplasmic constit- displays more ribosomes, thus becoming indistin- uents. The largest somas display far more neuro- guishable from granular endoplasmic reticulum. filaments which are often arrayed in loose bundles. The relationship between subsurface cisternae and Moreover, these aggregates create prominent granular reticulum is far less conspicuous than it "roads" in the cytoplasm ("Plasmastrassen," is in spinal cord cultures (26; or cord in situ, 39, Andres, 21), predominantly in the perinuclear 45). Also, the cultured ganglion subsurface cis- area, but also spreading peripherally where they ternae are less broad and less highly flattened than may seemingly divide the Nissl substance into dis- those in cultured spinal cord, in agreement with crete bodies (Fig. 7) (as in reference 34, also).

444 T~E JOURNAL OF CELL BIOLOGY • VOLUME32, 1967 Fibrillar areas or linear streaks lacking the normal rons, on the other hand, tend to occur singly, are fine cytoplasmic granularity have been observed more rounded, display a perinuclear zone in which in living neurons by a number of investigators; mitochondria, Golgi complexes, and dense bodies Pomerat et al. (14) have clearly demonstrated the are concentrated, and contain Nissl substance rapid movement of dense particles in these regions which is more concentrated in the periphery of the by means of time-lapse cinematography. Accom- perikaryon. Their most distinctive feature is the panying the neurofilaments are tubules similar to dilution of these cytoplasmic components by low the microtubules found in many mammalian cell density areas containing neurofilaments. These types, including neuronal somas (40-42, among are, therefore, the cells which other electron mi- others). Their outside diameter in our samples croscopists have labeled "light" (33, 37, 41) or averages 230 A. All neurons contain at least a few "Type A" (21, 43) as opposed to the small "dark" such tubules. They are often seen lying near and or "Type B" neurons. Andres (21) has further parallel to the plasma membrane. subdivided each type of neuron on the basis of In the preceding description, differences between nuclear properties, perikaryal size, predominance large and small neurons have been mentioned. The of "roads," axonal diameter and spiralling (glo- Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 smallest neurons tend to be grouped together, are merulus), and axonal ensheathment (myelin). The more angular in shape, contain Nissl substance small number of exiting neurites viewed and the dispersed throughout the perikaryon, and are wealth of transitional forms found militate against nearly devoid of neurofilaments. The largest neu- such elaborate classification in our preparations.

FIGURE 4 Variously sized and differently organized neurons (see text) are shown in this photomicrograph of a stained semi-thin section. As usual, the larger neurons are more rounded than the smaller neurons. A rarely seen axon hillock region is present in the binucleated neuron (n4). The largest neurons (e.g., n2) display lighter areas ("roads") in the cytoplasm. A "dunce cap" satellite cell is designated by an arrow. X 1,100.

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FIGURE 5 This electron micrograph portrays the small neurons in this culture system. Their organenes are quite evenly distributed throughout the cytoplasm, and neurofilaments are rare. A spinelike process emerges from the lower neuron at p. The neurons are completely covered by satellite cells. Invaginated into one of the satellite cells is an axon (ax) ; at higher magnification, a mesaxon can be followed from this axon to the satellite cell outer surface, gc ~ Golgi complex; n8 = Nissl substance. X 8,500.

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FmURB 6 Features of the large cultured neurons are shown in this electron mierograph. Mitoehondria, Golgi zones (gc), dense bodies, and slender bundles of neurofilaments occupy a wide perinuclear zone. Nissl substance (ns) is concentrated more peripherally. Satellite cell cytoplasm everywhere ensheathes the neuron surface. The aggregated gramflar material of unknown nature at x is frequently seen in cultured neurons. )< 8,500. Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021

FIGURE 7 This electron micrograph illustrates at higher magnification the type of cytoplasm characteris- tic of larger neurons. Mitochondria, Golgi complex (gc), Nissl substance (ns), dense bodies, neurofilaments (n]), a multivesicular body (mvb), and nuclear pores (at arrows) are included. A subsurface cisterna is indicated by an asterisk. Projections (pl-P3) from the neuronal soma, only one of which arises at this level (pl), are commonly found embedded in satellite cell cytoplasm. X ~0,000.

448 It appears to us that the larger the neuron, the were continuous with cisternae of granular endo- more prominent are the bundles of filaments and plasmic reticulum. Regarding mammalian dorsal resulting cytoplasmic partitioning. Andres reviews root ganglia, glycogen has thus far been described the literature suggesting a correlation between the as occurring normally only in embryonic stages neuronal types and different sensory modalities. (41, 46). The consistent variation in cytoplasmic organiza- The neuronal plasmalemma consistently appears tion found by electron microscopists should help more ruffled (Fig. 8) than the apposed plasma to resolve the long-standing question (35) con- membrane of the satellite cell, as indicated previ- cerning the role of faulty preservation in creating ously (21, 22). The neuron-satellite cell boundary is different types of dorsal root ganglion neurons. greatly complicated by a plethora of processes Single particles presumed to be glycogen are oc- from both the neuron and the satellite cell, as many casionally found throughout the cytoplasm or electron microscopists have documented (21, 37, grouped in sizeable aggregates most often pocketed 48-52). Slender evaginations of indeterminate just beneath the plasma membrane. These sphe- length arise from the perikaryon (large or small) roidal or irregularly shaped particles range from and invaginate attenuated or perinuclear portions ~190 to 350 A in diameter and stain intensely of satellite cells (Fig. 5) or extend along the gap with the lead solution employed, but not with between soma and satellite cell (P3, Fig. 7). These Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 aqueous uranyl acetate. In these properties, they "spines" do not completely penetrate the satellite correspond to particles identified as beta-glycogen cell sheath and, therefore, do not appose the base- in other tissues, as summarized by Revel (44). A ment membrane on the outer surface of the satellite few times, we have observed these particles to be cell. Cross-sections of similar appearing processes enmeshed in a large array of oriented membranous in which their connection to the soma is not present cisternae. This configuration resembles the "gly- are surrounded by a circle rather than a mesaxon cogen body" found in ethionine-treated liver by of satellite plasmalemma (P2, Fig. 7). Similar to Steiner et al. (63). Some of the involved cisternae neuronal processes demonstrated in electron mi-

FmURE 8 This neuronal satellite cell contains the usual cytoplasmic organelles, including cisternae of granular endoplasmic reticulum. A centriole (c) appears at this level. The chromatin pattern is charac- teristic for satellite ceils and Schwann cells in the cultures. The closely apposed satellite cell and neuron plasma membranes are indicated by arrows. X 16,500.

BUNGE, BUNGE, PETERSON, AND MURRAY Rat Dorsal Root Ganglion Cultures 449 croscopic studies of in vivo adult mammalian demarcated axon hillock region. This may be, in spinal ganglia (37, 48-50, 52, 53), these may rep- part, a reflection of the somewhat reduced size resent the pericellular and intracapsular network of neurons in the cultures, because Andres (21) of fine unmyelinated fibers (subcapsular den- rarely encountered hillock areas in the smaller drites?) observed in the light microscope (re- spinal ganglion neurons. The exiting axon may viewed in 49, 54). Additional neurite-like processes contain Nissl substance in its most proximal por- harbored within satellite cell cytoplasm differ tion, occasionally in the form of well oriented, from those just described in two ways. They are long cisternae of endoplasmic reticulum less larger in diameter than the slender somal spines densely covered with ribosomes and more shallow (but not so large as the main axon) and they are in depth than in the neighboring perikaryon. Such surrounded by an externally directed mesaxon configurations have been found in proximal den- (of satellite cell membrane), indicative of an ori- drites of spinal cord motor neurons by Bodian (45). gin outside the neuronal perikaryon. Are these In thin sections, evidence for a glomerulus is gen- branches of the exiting axon (recurrent collat- erally lacking; in only a few cases, two or three or erals?) or axons from other neurons? Rosenbluth more portions of the axon may be seen in one and Palay (50) found "axonal twigs" surrounded plane. This finding is in agreement with conclu- by a mesaxon of capsule cell membrane in rat dor- sions drawn from in vivo studies. Pannese (52) Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 sal root ganglia in vivo. In a study of dissociated points out that the glomerulus is rather uncommon chick spinal ganglion cells maintainted in vitro, in rat dorsal root ganglia and that those present Nakai (55) demonstrated the formation of collat- display relatively few coils. The glomerulus ap- eral processes which sometimes turned back to pears with increasing maturity of the tissue. Andres the region of the perikaryon. Whatever these proc- (2l) reports that only the larger dorsal root gan- esses may be, neither we nor anyone else have glion neurons display a well developed glomerulus. found electron microscopic evidence that they establish typical synaptic connections with the Satellite Cells soma. In the living culture, satellite cells are consistently In general, the main axon does not exit from a well associated with neuronal perikarya. The smallest

FIGURE 9 This photomicrograph illustrates the presence of reticu]in following Foot-Bielschowsky silver impregnation. The reticulin is oriented as a capsule around the neuron (n)-satellite cell complex and out- lines the nerve fibers in an emerging fascicle (]). X 1,050.

450 TEE JOURNAL OF CELL BIOLOGY - VOLUME 3~, 1967 Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021

FXGURE 10 Well organized nerve fiber bundles are shown in these stained semi-thin sections of the explant region and, in the inset, of the outgrowth (~0.5 em from the explant). Flattened perineurial cells (arrows) typically enclose a number of unmyelinated and myelinated fibers associated with Schwann ceils. The collagen substrate is at e. Figure, X 590; inset, X 1,150. somas are invested by two or three ceils, and as plasm of the fibroblasts. This is substantiated in many as ten to 12 may be found on the surface of electron micrographs--the arrangement of com- the largest neuronal cell bodies. ponents in satellite and Schwann cell nuclei ap- In semi-thin sections, no more than two satellite pears identical and contrasts with the more dis- cell nuclei are found at one level (Fig. 4). Some persed chromatin pattern in fibroblasts. nuclei are elliptical and sit on a flattened portion In the electron microscope, the satellite cell sheath of the neuronal soma in such a way that the re- is seen to cover the neuron completely (Figs. 5, 6). sulting topography conforms to the curvature of Substances outside the satellite cell must reach the the soma. Other nuclei are nearly triangular and neuron either by traversing the satellite cell or by perch atop the neuron like "dunce caps" (Fig. 4). moving through the small gap between neighbor- In addition, there are some nuclei which appear ing satellite cells, as earlier electron microscope perineuronal, but are actually separated from the studies implied (21, 37, 49, 51, 52). There is recent soma by basement membrane and collagen (as physiological evidence for substantial movement revealed by electron microscopy). Thus, they are of material through such narrow intercellular gaps related to the satellite cell-neuron complex, much in nervous tissue (reviewed in 56). The satellite as pericytes are situated on blood vessels. Nuclei cell layer is thin over much of the neuronal soma. are nearer the outer membrane of the satellite cell More than one layer may be present. The space than the inner one. Nucleoli are visible. The nu- between satellite cell and soma is fairly constant cleoplasm is more dense than that in the neuron. except where neuronal "spines" arise or groups of This density is comparable to that in Schwann cell entangled, slender satellite processes indent the nuclei and is definitely greater than in the nucleo- neuron (as in vivo, 50); here the space is uneven

BUNGE, BUNGE, PETERSON, AND M•UUAY Rat Dorsal Root Ganglion Cultures 451 (as in 21) and increased. Foci of intertwined satel- between apposed satellite cells. The number and lite cell processes are more often associated with complexity of evaginations from the outer surface the larger neurons. Whereas satellite cell processes of the satellite cell is greater in older cultures. The may cause shallow indentations in the neuronal pericyte-like satellite cell referred to earlier is profile, they do not invaginate the neuron in the separated from the perineuronal satellite cell by way that neuronal "spines" tunnel into satellite the two apposing basement membranes and inter- cell cytoplasm. vening collagen fibrils. It is not related to any Chromatin is more concentrated in the satellite nerve fibers. The difficulty one has in distinguish- cell nucleus than in the neuronal nucleus (as men- ing these from Schwann cells points to their mor- tioned in 37, 51, 53) and, in the best preserved phological similarity. Wyburn (49) calls them nuclei, clearly consists of two components. Near Schwann ceils. the nuclear envelope there is mainly fine-textured material whereas, more centrally, some nuclear Nerve Fiber Bundles components are larger and more dense, a pattern* In the living culture, bundles of nerve fibers are similar to that illustrated by Hay and Revel (57) seen to radiate out from the explant (Fig. 1). in interphase nuclei of regenerating salamander Schwann cell nuclei are spindle-shaped, whether Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 limbs. they be related to myefinated or unmyelinated The most striking feature of the perinuclear fibers (as in Fig. 1, reference 24), and on myeli- cytoplasm is the common occurrence of oriented nated fibers they are usually located midway be- cisternae of granular endoplasmic reticulum. Such tween nodes and are fitted into a curvature of the parallel arrays in situ have been observed by sheath, partially constricting the enclosed fiber. In Pannese (52), but not by others (21, 37, 49, 51, 53) these mature cultures, myelin sheaths are rarely The cisternae, sometimes branched, may be smooth; rippling and bleblike projections occur fre- densely covered by ribosomes and contain filamen- quently along the internode and may be further tous material. In some areas, cytoplasmic density is exaggerated into long, finger-like projections near increased by the close packing of cisternae of gran- the node (17, 59; as in Fig. l, reference 24). The ular endoplasmic reticulum and mitochondria. length of the internode averages ~ 128/~, varying Golgi complex consists of the usual vacuoles, cis- from 54 to 246 #. These internodes are substan- ternae, and vesicles. The lysosome-like bodies may tially shorter than their in vivo counterparts, as has be spotted with a more dense material. Multive- been pointed out in reference 30. Occasionally, sicular bodies, coated vesicles, centrioles, and an miniature internodes (~-~10/~ long) are observed, occasional filament are present. Microtubules are much as greatly shortened ("intercalated") inter- clearly visible and, particularly in the attenuated nodes are seen rarely in normal adult peripheral portions, follow the cell contour. Vesicles are nerve (60) and more often in regenerating nerve numerous, as earlier investigators found (21, 52, (61). Details of the frequently observed Schmidt- 58), but may be nearly twice as large (600-1300 A) as their in vivo counterparts; they are more often Lanterman clefts, which form and may undergo lined up near the membrane facing the neuronal alterations during in vitro existence, appear else- soma rather than the outer membrane, in agree- where (1, 17, 62). Since the clefts are found in ment with observations made in vivo (21, 52). living cultures, they cannot be considered to result Some of these appear coated. Many vacuoles of from preparative procedures (1). In the polarizing varying size are seen. Some of the larger vacuoles microscope internodes are birefringent (1; as in and the often widened nuclear envelope and cis- Fig. 8, reference 24). Areas between fascicles con- ternae of granular endoplasmic reticulum probably tain characteristically flattened fibroblasts. During result from the preparative procedure. the first few days of culture, capillaries migrate The outer plasma membrane is smoother than from the explant in both solid and tubular forms; the membrane facing the neuronal soma and is in time, they lose their distinctive organization everywhere covered by a basement membrane. and may no longer be recognizable. This basement membrane closely follows the cell In the whole mount stained with Sudan black, the contour, including the slender processes (or flat entire internode is sudanophilic and the myelin sheets?) of cytoplasm emanating from the satellite features just described are confirmed. By compar- cells, but typically does not extend into the gap ing cultures before and after OsO4 fixation and

452 ThE JOURNAL OF CELL BIOLOGY " VOLUME 3~, 1967 Sudan black staining, it was observed that this In semi-thin sections, a fascicle (designated by type of preparation faithfully preserves myelin arrows in Fig. 10) is demarcated by a complete or irregularities and causes little shrinkage; i.e., in- partial sheath of perineurium and usually contains, ternodal length is decreased by less than 2%. regardless of the number of fibers, a few myelinated Branching of the myelinated neurites is observed, and many more unmyelinated fibers plus associ- always at a node of Ranvier. With the Holmes' ated Schwann cells. Following thorough acquain- silver method, nerve fibers are seen to terminate as tance with the living cultures, it was surprising to a simple spray of branches with bouton swellings find such high proportions of unmyelinated fibers among fibroblasts in the outgrowth. Functional in heavily myelinated cultures both in these prep- aspects of these terminals are not known but it may arations and in electron micrographs. The peri- be noted that similar appearing endings on muscle neurial cells are greatly flattened and display nu- ribbons in other preparations participate in reflex clei which are lighter than those in nearby arc activity (8; E. R. Peterson and S. M. Crain, Schwann cells. The seemingly clear areas evi- unpublished observations). With the Foot-Biel- ent in Fig. 10 will be shown below to contain endo- schowsky silver stain, thin black fibrous sheaths neurial components. are present in fascicle areas and in capsules around In the electron microscope, a typical fascicle is seen neurons, indicating the widespread occurrence of to consist of a perineurial sheath partially or com- Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 reticulin (Fig. 9). pletely enclosing Schwann cells and their mye-

FIGURE 11 The components of a nerve fiber bundle are depicted in this electron micrograph. Enclosed by the flattened perineurial cells (pc) are Schwann cells and their related unInyelinated and myelinated fibers. Note that the myelinated fibers occur singly in a Schwann cell. The two pairs of arrows indicate the span of regions of close contact between perineurial cells (see text). X %000.

BUNG-E, BUNGE, PETERSON, AND MURRAY Rat Dorsal Root Ganglion Cultures 4,53 Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021

FmURE 1~ The Schwann cell-nerve fiber relationship, basement membrane (bin), and collageu fibrils (c') are illustrated in this electron micrograph. Inner or outer mesaxons appear at the black arrows. White arrows point to microtubulcs in Schwann cell cytoplasm. A Schwann cell multivesicular body with a plaque of fuzzy substance on its exterior is seen at x. X ~5,000.

454 THE JOURNAL OF CELL BIOLOGY • VOLUME 8~, 1967 linated and unmyelinated fibers, basement mem- tance (and may contain collagen), or is diminished, brane, and collagen fibrils (Figs. 11, 12). This ar- thereby allowing closer cell contact. These regions chitecture is present in the explant and also in the of close contact, commonly seen in vivo (64, 65), outgrowth zone which is organized entirely in may be of considerable length (referred to in Fig. vitro. The perineuriurn is composed of concentric 1 I) and average ~180 A from one cytoplasmic layers (one or usually four or five or sometimes surface to the other. In perineurial cell cytoplasm, more) of interdigitating t~attened cells. The usual long cisternae of granular endoplasmic reticulum intercellular gap between these layers is in some and small vesicles are prominent and particles places widened locally (Fig. 11) or for some dis- presumed to be glycogen are often seen scattered Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021

FIGURE 18 This figure demonstrates the number of unmyelinated axons that may be related to one Schwann cell. Most of the axons are completely invested, resulting in a typical mesaxon configuration (arrows). The basement membrane covering of the Schwann cell and collagen fibrils (c) are also illustrated. X el,000.

FmtmES 14 and 15 Centrioles and associated cilia are shown in these portions of Schwann cell cytoplasm. (An identifying axon (ax) is present in one area.) In Fig. 14, the centriole is best seen; in it are the typical peripheral triplets of tubule-like dements. In Fig. 15, the cilium and surrounding ciliary vesicle are better shown. It is common to see centrioles and cilia in this relationship in cultured Schwann cells. Fig. 14, X ~8,500; Fig. 15, X ~3,000.

BUNGE, BUNGE, PETERSON, AND MURR&Y Rat Dorsal Root Ganglion Cultures 455 throughout or appear in aggregates. A perineurial areas facing the cytocentrum. The cytoplasm con- sheath is covered internally and externally by tains granular endoplasmic reticulum in the form basement membranes, but many of the interme- of scattered long, sometimes branched, widened diate layers of this sheath are not, as Waggener cisternae which contain filamentous material and et al. (66) have observed in rat sciatic nerve. The are regularly and densely covered with ribosomes. fine structure of the perlneurium in culture closely An occasional cisterna may be greatly distended resembles that described for mammalian periph- by accumulations of contained substance. Multi- eral nerve in situ by Thomas (64) and Gamble vesicular bodies resemble those in neurons, includ- (65), among others. Within the perineurium, an ing the plaque of dense substance adhering to the infrequent fibroblast or histiocyte may be found. outer surface. Vesicles of varying size are numer- The fibroblast is here identified by its lack of base- ous. An occasional filament is seen. Filaments have ment membrane, lighter cytoplasm compared to been noted in the Schwann cells of cat splenic the Schwann cell, prominent granular endoplas- nerve (67). Microtubules averaging 230 A are mic reticulum which is often oriented in parallel present throughout Schwann cell cytoplasm. They arrays and filled with filamentous material, large are particularly noticeable in the cytoplasmic vacuoles bounded by agranular membrane, and

collar inside and outside the myelin sheath where Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 bundles of filaments in the cytoplasm. These cells they lie parallel to the fiber length (Figs. 16-18, 20). are triangular or crescentic with elongated proc- In the paranodal cytoplasmic spiral and in cleft esses. This description of fibroblasts fits that cytoplasm, however, they curve around the axis presented by others (64, 65) for peripheral nerve cylinder. Thus, when the fiber is sectioned longi- and dorsal root in situ. The histiocyte is distin- tudinally, cross- or obliquely sectioned tubules are guished from the other cell types by its ruffled, seen in the paranodal (Fig. 20) or cleft (Fig. 19) oudine, which often gives rise to slender processes, cytoplasm whereas, when the fiber is sectioned its lack of basement membrane, predominance of transversely, tubules appear in longitudinal section vesicles and vacuoles, paucity of granular endo- following the curve of terminating cytoplasm. The plasmic retieulum, and an occasional heterogene- tubules are also found in Schwann cells investing ous appearing inclusion body or multivesicular unmyelinated fibers. Sandborn et al. (42) have body that is larger and more irregularly shaped reported the presence of microtubules in Schwann than elsewhere in the culture. cells related to rat sympathetic nerve fibers. The Schwann cell nucleus is composed of two dif- Centrioles and cilia are encountered occasionally ferently appearing chromatin components (Figs. (Figs. 14, 15) but only in Schwann cells related to 12, 13) as in the satellite cell nucleus. Evenly unmyelinated fibers. This is in contrast to a claim spaced particles are arrayed on the inner mem- made by Barton (68) that centrioles are present brane of the nuclear envelope. The gap between only in myelin-related Schwann cells but is in the inner and outer membranes varies because the agreement with a conclusion reached by Grillo outer membrane is irregularly wavy and may form and Palay (69). The cilium is usually seen to infrequent outpocketings containing dense ma- arise from one of a pair of centrioles and contains terial. Ribosomes are regularly arrayed on certain a large central vesicle, a distinctive feature of areas of the outer membrane, particularly those Schwanv cells (69). Other morphological differ-

FIOU[tE 16 This electron micrograph illustrates a myelinated fiber (ax) and investing Schwann cell in longitudinal section. Lengths of Schwann cell microtubules are seen run- ning parallel to the fiber (and may be compared to the axoplasmic filaments). Collagen fibrils (c) have also been sectioned longitudinally. × $8,000.

FmVEE 17 Microtubules appear as circles when the Schwann cell and related fiber are transversely sectioned. Cross-sectioned neurofilaments are present in the axon (ax). An outer mesaxon is also shown (arrow).)< 45,000.

FmuaE 18 This electron micrograph illustrates the similarity of myelin formed in vitro to that observed in vivo (see text). Mesaxons are indicated by arrows. Microtubules are present in the Schwann cell cytoplasm internal and external to the sheath. X ll0,000.

456 THE JOURNAL OF CELL BIOLOGY • VOLUME32, 1967 Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021

BUNGE, BUNGE, PETERSON, AND MURRAY Rat Dorsal Root Ganglion Cultures 4,57 an irregular, oversized envelope (Fig. 12). Gamble (65) mentions the very occasional presence of bleblike projections of basement membrane in adult rat dorsal root. The proportion of folded projections observed in the cultures is probably more than that found normally after maturation has occurred. Highly folded basement membranes are common in degenerating dorsal root (70-72) in which they are thought to result from loss in volume of contained structures; i.e., loss of axons. Occasionally, multiple layers of basement membrane, many of them incomplete, surround a Schwann cell. Within these layers an occasional collagen fibril may be detected (Fig. 12). Gamble (65) found multiple basement membrane layers and bundles of collagen among these layers in adult rat sural nerve and, to a lesser degree, in rat dorsal root. Collagen fibrils were found frequently Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 among multiple laminae in regenerating rat dorsal roots by Nathaniel and Pease (72) who used this and additional evidence to suggest that the Schwann cell can both form and polymerize tro- pocollagen. Light microscopic indication of reticulin formation by Schwann cells in vitro has been FIOURE 19 A Schmidt-Lanterman cleft appears offered by Peterson and Murray who found that in this electron micrograph. At the arrows, the the pattern of newly formed reticulin fibers con- components of the major dense line separate and forms to Schwann cell orientation (5), depends enclose Schwann cell cytoplasm. Within this cyto- upon the nutritional status of the Schwann cell plasm of the longitudinally sectioned sheath, transversely sectioned microtubules are present (near (97), and does not exhibit a spatial relationship to arrows). The position of these circular profiles sug- adventitious fibroblasts or other cells. gests that they belong to the same microtubule. Nerve fibers typically contain neurofilaments, )< 31,500. various tubular components, mitochondria, and vesicles or vacuoles of varying size (Fig. 20). Mito- chondrial cristae most often lie in a plane perpen- ences between the cytoplasm of Schwann cells with dicular to the length of the mitochondrion. Vacu- unmyelinated fibers and that of Schwann cells oles are frequently situated close to the axolemma. with myelinated fibers were not apparent. Particles presumed to be glycogen are observed Schwann cells are typically coated with basement membrane (Figs. 12-18). Usually there is but one infrequently. According to random measurements layer and it closely follows the cell contour, also obtained from light and electron micrographs, covering the nerve fibers where they are not com- fiber diameter measures from 0.2 to 2.6 ~ for the pletely encircled by Schwann cell cytoplasm. axon itself, or up to 4 # if the myelin sheath is in- Occasionally, the basement membrane veers away- cluded. Diameters of fiber plus myelin may reach from the Schwann cell in folds, thus appearing as 13 /~ in dorsal roots of 6-month-old rats (77). The

FIGURE ~0 This electron micrograph shows one of the nodes found regularly along myelinated fibers in the cultured ganglia. Typically, the myelin lamellae terminate in loops, the innermost loop occurring farthest from the node. Microtubules are present in the Schwann cell cytoplasm outside the sheath and inside a loop (arrows). Near the node is a long slender projection of myelin which is seen to be continuous with the lower myelin sheath at another level. Schwann cell processes with their overlying basement membrane interdigitate at the node. )< ~7 500.

458 T~E JOURNAL OF CELL BIOLOGY " VOLUME 3~2, 1967 Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021

BUNGE, BUNGE, PETERSON, AND MURRAY Rat Dorsal Root Ganglion Cultures 459 parameters of myelinated nerve fiber diameter as mesaxons are shown in Figs. 12, 17, and 18. Occa- well as internode length and neuronal soma size sionally, an axon is surrounded by many turns of (described briefly in reference 30) will be con- a spiraled mesaxon which probably would have sidered in more detail in a future publication. The been compacted during further maturation. A few smaller fibers are invested with cytoplasm of turns of Schwann cell cytoplasm around an axon Schwann cells which form the well known are noted frequently; some myelination is un- mesaxons (Fig. 13) (73, 74). The larger neurites oc- doubtedly continuing at a month or more in vitro. cur singly in a Schwann cell and are usually myeli- Schmidt-Lanterman clefts are visible in both nated as in situ (74, 75). Myelinated fibers always transverse and longitudinal (Fig. 19) sections. As occur singly in a Schwann cell (Figs. 11, 12). An described earlier (82), a cleft is a series of splits in occasional configuration resembling a second mye- myelin lamellae at the major dense line with por~ linated fiber is seen, upon closer inspection, to lack tions of Schwann cell cytoplasm enclosed, and it an axon and to be, therefore, a myelin evagination. may be regular, as pictured in the figure, or a more The occurrence of multiple unmyelinated or single complex and irregular structure. Nodes of Ranvier myelinated fibers within a Schwann cell has been are covered by interdigitating Schwann cell proc- reported already for cultured dorsal root ganglia esses within a continuous basement membrane, Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 (19). The critical size for myelination in the culture and the paranodal myelin terminates in loops, the is .~ 1 ~, i.e., in electron micrographs fewfibers be- loop nearest the node arising from the outermost low 1 /~ in diameter are myelinated whereas most myelin unit (Fig. 20) (as in situ 83-85). In addi- fibers greater than 1 # display myelin sheaths (30). tion to tubules, many dense particles are found Gasser (73) found that cat dorsal root unmyelin- within the terminating loops and, as Elfvin (85) ated fibers in electron micrographs generally meas- points out, the particles are often clustered on the ured 0.15-0.5 /~, but trailed off to 1.0 ~. In a membrane adjacent to the axolemma and at times study of sensory nerves in mouse cornea, Whitear resemble ribosomes. Evenly spaced particles dot (76) found that fibers measured 0.15 to just over the gap between some terminating Schwann loops 2 # in electron micrographs; in some graphs of and axolemma in a fashion similar to that shown these data, it appeared that myelination occurred so clearly in hedgehog nerve by Bargmann and as the fibers approached 1 # in diameter. Peters Lindner (86). In a few cases, we have noted a and Muir (75) state that, in developing peripheral stack of desmosome-like densities (including the nerve, axons undergoing myelination measure locally widened intercellular gaps) between neigh- 2-4/z whereas unmyelinated grouped axons are 1 boring regions of Schwann cell cytoplasm in node or less in diameter. From a light microscope and cleft areas; these configurations resemble study of mammalian dorsal and ventral roots, those shown among paranodal loops in rat vestib- Duncan (77) surmised that the critical size for ular ganglion (87) and rat sciatic nerve (88). myelination was around 1 /~. In rare cases in the Especially at the node and occasionally elsewhere cultured ganglion, a fiber may contain an accumu- along the sheath, long outpocketings of myelin lie lation of pleomorphic dense bodies displaying areas parallel to the sheath length (Fig. 20). We con- resembling myelin figures, interpreted as a sign of sider these loops to be continuous with the sheath degeneration (23, 78; see also 79). A rare fiber (de- (though they may appear not to be, because of the teriorating?, reference 23) contains mitochondria plane of thin sectioning) and to be devoid of axn- which have rounded up from their usual long plasm. In cross-section, local bulges and loops are slender shape, with granular substance rather than commonly seen and some sheaths appear larger cristae mitochondriales occupying the central than their contained axons, resulting in comma- or region. Although the dorsal root ganglion culture banjo-like (Fig. 12) configurations. These irregu- is a fairly stable population, from time to time larities are not preparative artifacts because they neurons may degenerate for no apparent reason. are observed in living cultures; nor are variations Myelin sheaths display the typical lamellated produced solely as a result of in vitro existence structure with a repeating period of ~120-150 A for myelin loops and folds, including long out- (Fig. 18), a range equaling that for in vivo periph- pocketings near nodes, exist in mammalian peripheral nerve (47, 80, 81) after OsO4 fixation and eral nerve in situ (80). epoxy resin embedding. The cultured myelin Endoneurial collagen measures 200-450 A in sheaths vary little in thickness. The inner and outer diameter as compared to measurements of 250-

460 THE JOURNAL OF CELL BIOLOGY • VOLUME 3~, 1967 550 A (65) or 300-500 A (21) in rat dorsal root in connective tissue elements, and associated peri- vivo. The distribution of this collagen ranges from neurial constituents. Organization of the peri- an uneven sparse to a more uniform, fairly dense neurium is attained even though the fibers grow accumulation always associated with a mottled out on a flat surface; this is in contrast to the view background suggesting the presence of additional held by Nakai (cultured chick ganglia, 91), who matrix substance. Generally, these fibrils are nei- claims that a three-dimensional network is nec- ther gathered into bundles nor as closely com- essary for development of perineurial elements in pacted as they appear to be in peripheral nerve in vitro. vivo (64). Furthermore, they are not condensed The problem of what may influence the course into a continuous sheath around the nerve fiber, of outgrowing nerve fibers has been extensively and the collagen present is not organized into two studied in recent decades (93). The fascicle forma- layers, the inner and outer endoneurial sheaths tion that occurs in the ganglion cultures grown on (Figs. 12, 20). These findings agree with Gamble's a two-dimensional collagen substrate may appear observations of rat dorsal root in situ (65) showing at first glance difficult to explain on the basis of that, in contrast to more distal peripheral nerve, concepts such as contact guidance and selective the amount of collagen is smaller and is not organ- fasciculation. The relationship between connective Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 ized into two endoneurial sheaths. The collagen tissue and nerve fibers may be a critical factor in associated with cultured fibers shows some char- the cultures described in the present study. When acteristics of the inner endoneurial sheath (or the well encapsulated ganglia are explanted, nerve sheath of Plenk and Laidlaw, reference 89); i.e., fibers emerge as stout fascicles, primarily at the though most fibrils run parallel to nerve fiber central and peripheral roots but also at other length, there is some variation in their orientation, points where the capsule has weakened and can be the fibrils are not densely packed, and the presence penetrated. When the capsular investment is of intervening matrix is indicated. As Thomas (89) minimal (due to rupture or deliberate removal or summarizes, it is the inner endoneurial sheath that because the ganglion is taken at a very early fetal is the argyrophilic or reticulin layer, the reticulin age), nerve fibers emerge without becoming asso- staining probably depending on the associated ciated either as an even radial outgrowth or as a carbohydrate in the matrix. This, then, would ex- loose tangle of unoriented elements. The nonag- plain why reticulin rather than collagen staining gregated fibers do, however, often become grouped is heavy in the cultures (Fig. 9). The fibrils per se into bundles in more distal regions of the out- cannot be identified as collagen or reticulin com- growth (see Fig. 1). ponents electron microscopically (90). Some of the Whereas the presence of capsular tissue would collagen fibrils found in the explant have been help to explain the formation of primary fascicles carried into the culture at explantation but many near the explant, the factors operating in the more and particularly those in the outgrowth form in distal regions to induce fascicle formation are less vitro. clear. In addition to the partially ruptured capsule, vestiges of meninges are commonly explanted with FURTHER COMMENTS the ganglia (unless deliberately removed by pains- taking dissection). Both of these connective tissue Fascicle Formation elements migrate and proliferate rapidly over the The gathering of lengthening nerve fibers into collagen surface and soon outstrip the early burst histiotypic fascicles, cven in distal regions of the of neuritic outgrowth. This modifies the surface of outgrowth, is one of the most striking aspects of the collagen in areas extending far from the thc cultured rat dorsal root ganglion (Fig. 10). original explant by introducing inhomogeneities This organization of the outgrowing fibers involves which may mark out linear paths for the outgrow- both the convergence of fibers into bundles (fascicle ing nerve fiber. Thus the neurites may be guided formation) and the cxchangc of nerve fibers by contact with this connective tissue terrain. between bundles (plexus formation). The final re- Weiss (98), under the general term of "contact sult is a series of intcrconncctcd fasciclcs separated guidance," has repeatedly stressed the importance by largc fields of connective tissue devoid of nerve of the orientation of the components of the sub- fibers (Fig. 1). These fascicles contain both mye- strate or surrounding matrix in the determination linated and unmyelinated fibers, Schwann cells, of the course taken by nerve fibers in vitro.

BUNGE, BUNGE, PETERSON, AND MURRAY Rat Dorsal Root Ganglion Cultures 461 Other factors influencing the formation of nerve portion of cytoplasmic to nuclear mass being fascicles have been proposed. Based primarily on greater. During Nissl development, an inter- in vivo experiments (92, 93), it has been suggested mediate pattern of large peripheral masses and that pioneering fibers, upon reaching their periph- small central particles is observed in chick but not eral termination, become endowed with a selective in rat cultured neurons. The Nissl bodies of ma- adhesiveness which draws subsequent outgrowing tured rat neurons are smaller than those in ma- fibers into the same pathway. Weiss (93) concludes tured chick neurons. An abundance of neuro- that this "selective fasciculation" occurs only if fibrils is readily demonstrated, in chick neurons, the pioneering fiber finds adequate peripheral with Bodian's and Holmes' silver stains; a fine termination. The extensive fascicle formation de- neurofibrillar network is seen in rat neurons only scribed in the present paper would appear to con- after Holmes' stain. Rat ganglion neurons, in gen- tradict this contention unless the simple endings eral, display more associated satellite cells than found in our ganglion cultures can be considered the usual two to four satellite cells investing chick adequate peripheral termination. The physiology neurons. In the chick culture, well developed retic- of sensory nerve endings in culture is not known. ulin sheaths are demonstrable by the 2nd wk in Although Lumsden (94) clearly illustrated vitro (prior to myelination), whereas in rat cultures the presence of nerve endings in cultures of chick more delicate reticulin sheaths usually appear after Downloaded from http://rupress.org/jcb/article-pdf/32/2/439/1263268/439.pdf by guest on 01 October 2021 dorsal root ganglia, he preferred to consider that myelination has begun. The period of myelination fascicle formation is controlled by forces within is similar in both culture systems, but myelin for- the neuron rather than by influences dependent mation is far more extensive in the rat tissue; nerve upon neuritic termination. Another factor of pos- fibers from the rat neurons are usually continuously sible significance in fascicle formation is the forma- enveloped by myelin segments far into the out- tion of filopodia (from the growth cone or nearby growth, whereas chick fibers more often display lateral regions of the fiber) which seek out and shorter groupings of internodes, or even isolated sometimes adhere to similar projections from other segments (99). Rat myelin is readily stained by the fibers, subsequently pulling the nerve fibers to- standard Luxol fast blue technique; chick myelin gether as they retract (95, 96). Much remains to is stained by this method only after chromation. be learned of the influences on nerve fiber course Finally, fascicle formation is more prominent in and organization; the special contribution of the rat ganglion cultures than in chick ganglion cul- present study is the demonstration of the complex tures. It is not known whether this is influenced by three-dimensional organization which may occur one obvious difference in the two types of ganglia, in cultured nerve tissue, an organization involving i.e., the presence on rat ganglia of meningeal rem- the hisfiotypic interrelationship of connective tis- nants which are absent from chick ganglia (be- sue, Schwann cell, and nerve fiber elements. cause of their location outside the vertebral column). Additional details and illustrations of Rat vs. Chick Cultures points of difference between rat and chick cultures In vitro development of rat dorsal root ganglia appear in reference 16. differs in a number of ways from that of the chick dorsal root ganglia described earlier (5). The We wish to express our sincere appreciation to Dr. study of chick ganglion cultures was done with a George D. Pappas in whose laboratory M.B.B. was feeding medium containing --~100 mg % glucose; a Postdoctoral Fellow when this project was initiated. higher levels of glucose do not appear to enhance The valuable technical assistance of Mrs. Joeline development. This amount of glucose will foster Spivack, Mr. Philip Chang, and Mr. Vincent Blan- neuronal maturation in rat ganglion cultures, but cuzzi is gratefully acknowledged. myelin formation is greatly enhanced if the glucose This study was supported by National Institutes of Health Grant NB 04235 and National Multiple level is raised to 600 mg %. The collagen substrate Sclerosis Society Grant 328 to Dr. R. P. Bunge, so useful for long-term culture of rat ganglia is not National Institutes of Health Grant NB 00858 to sufficient for the maintenance of chick ganglia be~ Dr. M. R. Murray, and National Institutes of yond the 4th wk in vitro. If the collagen is supple- Health Grant 5TI-GM-256 to Dr. W. Copenhaver. mented with a plasma clot during the 3rd wk, the A preliminary report of this work was presented otherwise imminent acute degeneration is pre- at the 2nd Annual Meeting of the American Society vented. Mature neurons in cultured chick ganglia for Cell Biology, San Francisco, 1962 (20). are larger than those in rat cultures, with the pro- Received for publication 5 July 1966.

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466 THE JOURNAL OF CELL BIOLOGY • VOLUME3~, 1967