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Neuron-schwann cell interaction in basal lamina formation
DEVELOPMENTAL
BIOLOGY
g&449-460
(19%)
Neuron-Schwann Department
Cell Interaction
in Basal Lamina Formation
MARY BARTLETTBUNGE,'ANN K.
WILLIAMS,
of Anatomy
University
and Neurobiology,
Washington
AND PATRICK School of Medicine,
Received December 10, 1981; accepted in revised fwm
M. WOOD St. Louis, Missouri
63110
March 29, 1982
The availability of tissue culture systems that allow the growth of nerve cells, Schwann cells, and fibroblasts separately or in various combinations now makes possible investigation of the role of cell interactions in the development of the peripheral nervous system. Using these systems it was earlier found that basal lamina is formed on the Schwann cell surface in cultures of sensory ganglion cells and Schwann cells without fibroblasts. It is here reported that the presence of nerve cells is required for the generation of basal lamina on the Schwann cell plasmalemma. Utilizing nerve cell-Schwann cell preparations devoid of fibroblasts, this was found in the following ways. (1) When nerve cells are removed from 3- to 5-week-old cultures, the basal lamina disappears from Schwann cells. (2) If nerve cells are added back to such Schwann cell populations, Schwann cell basal lamina reappears. (3) Removal of nerve cells from older (3-4 months) cultures does not lead to basal lamina loss; areas presumed not to have been coated with lamina before neurite degeneration remain so, suggesting that the lamina persists but is not reformed. (4) If basal lamina is removed with trypsin, it is reformed in neuron plus Schwann cell cultures but not in Schwann cell populations alone. Thus, the formation but not the persistence of Schwann cell basal lamina requires the presence of nerve cells. INTRODUCTION
This paper is one of a series describing tissue culture studies designed to elucidate the role of cell interactions in the development of the peripheral nervous system. Methods are now available for culturing rat sensory ganglion nerve cells, (NCs),’ Schwann cells (SCs) and several types of fibroblasts either separately or in various combinations (Wood, 1976; Bunge et al, 1977). The initial paper in this series compared cultures containing NCs alone with cultures containing both NCs and SCs (Bunge et al., 1980). In spite of the lack of cellular ensheathment, cultured sensory neurons bereft of other cell types develop and maintain a dense outgrowth of nerve fiber fascicles for many months. Basal lamina and collagen fibrils are not formed. When SCs accompany the neurons, nerve fibers are ensheathed as they are in situ, with either SC cytoplasm or myelin, and basal lamina (BL) and thin collagenous fibrils are generated around the SCs. In this report we present evidence that interaction between NCs and SCs is required for BL formation on the SC. This evidence has been obtained by (1) removing NCs from NC + SC cultures, with the ensuing loss of BL from the SC surface (if the cultures are of a relatively young age); (2) reintroducing NCs into cultures containing only SCs, with the subsequent reappearance of BL; and (3) comparing regeneration of BL in cultures of NCs + SCs or SCs alone after removal i To whom all correspondence should be addressed. ‘Abbreviations used: NC, nerve cell; SC, Schwann cell; BL, basal lamina; BSS, balanced salt solution.
of the BL by means of trypsin treatment. Some of these findings have been presented in preliminary form (Williams et ab, 1976; Bunge et al., 1979). MATERIALS
AND METHODS
The culture and preparatory techniques for light and electron microscopy have been published previously (Wood, 1976; Bunge et al., 1980). In brief, 17- to 21-day fetal or newborn rat (Holtzman) dorsal root ganglia were placed on a reconstituted rat tail tendon collagen substratum and fed a complex medium containing nerve growth factor. The reader is referred to the references cited above for the regimen devised to obtain NC or NC + SC cultures free of fibroblasts by the use of the antimitotic agents, cytosine arabinoside and fluorodeoxyuridine. After the antimitotic treatment, ganglia were transplanted to allow the formation of a new outgrowth in the absence of these agents. To obtain SC preparations from NC + SC cultures, the explant containing all the neuronal somata was cut out, thereby causing the demise of all nerve fibers within a few days. Carefully prepared explants essentially containing only NCs could be subsequently transplanted into cultures consisting only of SCs. It was the outgrowth area that was generated entirely in vitro that was examined for this study. Cultures were maintained for periods of a few weeks up to several months. To study the effect of neuronal loss, SC cultures were examined 1, 2, 3, 4, 7, 8, 13, 14, 41, or 60 days after extirpation of the explant from NC + SC cultures of about 1 month of in vitro life. These
449 0012-1606/82/080449-12$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
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experiments utilized five culture series. Also, neurons were removed from 3- or 4-month-old NC + SC cultures and these cultures were maintained for an additional 2- or &week period. Treated explants composed of NCs alone were added to SC preparations that had been divested of neurons previously; these cultures were examined 3 or 5 weeks later. Trypsin treatment consisted of first rinsing the NC + SC or SC culture three times in Ca-Mg-free Hanks’ balanced salt solution (BSS), incubating in 0.25% trypsin (3X crystallized, Worthington Biochemical Corp., Freehold, N. J.) in the same fluid for 1 hr at 35°C and rinsing in Earle’s BSS before fixation or return to the usual culture medium. NC + SC or SC cultures (3 or 6% weeks or 4 months old) were studied directly after trypsin treatment or 1, 3, 7, 14, 16, or 30 days later. Three series of cultures were used for this part of the study. Cultures were fixed initially in arsenate-buffered 2% glutaraldehyde with 1% sucrose after prefatory rinsing in warmed Earle’s BSS, rinsed in BSS, further fixed in 2% 0~0~ in BSS, rinsed again in BSS, sometimes stained en bloc with uranyl acetate, dehydrated in graded ethanol and propylene oxide, and embedded in Epon-Araldite (as in Bunge et al., 1980). Suitable areas in the embedded specimen were located by phase microscopy; care was taken to sample outgrowth areas that were a similar distance from the explant, and thus comparable in maturation. These were sectioned with a diamond knife in an LKB-Huxley ultramicrotome, placed on carbon-stabilized Formvar-coated grids, stained with uranyl acetate or tannic acid (Bunge et al., 1980) and lead citrate, and examined in a Philips 300 electron microscope. RESULTS
These cultures consist of a central explant containing cell bodies of sensory neurons surrounded by a halo of neurite-SC outgrowth (free of fibroblasts) several millimeters in diameter. The morphology of the NC + SC culture outgrowth has been described previously (Bunge et al., 1980). The findings germane to this paper are that cultured SCs ensheathe nerve fibers in the normal way; one SC is related to a number of smaller unmyelinated neurites or to a single larger myelinated axon. The neu-
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rite-SC partners are enveloped by BL which thickens with increasing time in culture. The lamina coats those areas of the SC surface that interface with the extracellular milieu; there are some “intramural” SC processes as well as areas of the SC surface apposed to neurites that lack BL. Discrete regions of the SC surface membrane exhibit subplasmalemmal density which in combination with apposed BL resembles a small hemidesmosome. Scattered bundles of slender collagenous fibrils (mean diameter, 18 nm) may be observed in the extracellular space, usually in association with the lamina and situated parallel to the longitudinal axis of the fascicles (Fig. 1). Neuron removal. When the explant containing the neuronal somata is cut out of NC + SC cultures, outlying neurites degenerate rapidly; deteriorating axons are seen at 1 and 2 days but neurites are no longer identifiable at 3 days after neuron removal. In these early time intervals following explant extirpation, SC lysosomal dense bodies become enlarged and more heterogeneous in content and multivesicular bodies increase in number. Basal lamina is still visible on portions of the SC surface (Fig. 2). During these early periods before BL disappearance, the lamina usually is found on SC plasmalemmal areas lying in apposition to the extracellular fibrils which persist (as in Fig. 3); this indicates that these surface domains had bordered the extracellular space instead of interfacing with neurites or intramural SC surfaces that lack basal lamina when neurites are present. Thus, it is concluded that in the absence of neurons BL persists for a few days but no new lamina is being formed on areas that had formerly been devoid of it. Although an occasional patch may be observed, most of the BL has disappeared by 7 or 8 days following NC removal from these relatively young cultures of about 1 month in vitro age (Fig. 3). An occasional cistern of granular endoplasmic reticulum is seen to be engorged with sequestered material, although most cisterns appear normal in width and content. Basal lamina does not reappear in sibling cultures divested of NCs for 6 or 8 weeks (Fig. 4). The time course for BL disappearance from monthold cultures was consistent from experiment to experiment with one exception. In one series of cultures, ex-
FIG. 1. Control NC + SC culture. Schwann cell cytoplasm associated with neurites (n) is covered by BL (b). External to the BL collagenous fibrils that have been cross-sectioned. This and all other figures illustrate areas of transversely sectioned outgrowth formed in vitro and did not contain fibroblasts. Three weeks in vitro; x27.000. FIG. 2. Three days after NC removal from a NC + SC preparation in the same culture series as Fig. 1. Basal lamina is visible (see ,) but not all areas of the SC surface. The neurites degenerate within this time period. X38,000. FIG. 3. Seven days following NC removal from another sibling culture. The BL has vanished except for an occasional thinned (b) that is situated near collagen fibrils. Rough endoplasmic reticulum is prominent in one area of SC cytoplasm (at the micrograph ~36,000.
are thin that was on some segment center).
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amined 1, 3, 7, and 14 days after neuron removal, patches of BL were still evident in the 14-day sample. A notable difference between this culture series and the others was the presence of a greater than usual number of collagenous fibrils (see Fig. 9 in Bunge et al., 1980), suggesting that there had been greater secretory activity in this series than in the others studied. Enhanced secretory activity may have led to the formation of a more stable lamina in this culture period. This interpretation is consistent with results obtained from older cultures. When neurons are removed from 3- or 4-month-old NC + SC cultures, BL is still present 8 weeks later (Fig. 5). The lamina was undoubtedly more stable at these later times of NC removal because we have observed that it becomes more continuous and increases in thickness with time in culture (Bunge et al., 1980). At 3 or 4 weeks in culture, the lamina may be thin and discontinuous in contrast to that of older cultures. As noted after neuron removal from younger cultures, when basal laminae are present 8 weeks after neuronal loss from 3- or 4-month-old cultures, they are observed only on certain regions of the SC surface. The interpretation that these patches contain persisting rather than reforming lamina material in the absence of accompanying NCs prompted the trypsin experiments described in the next section. In spite of the lack of neurite contact for periods up to 4 weeks, SCs remain in chains of elongated cells on the collagen substratum and do not migrate from these extended arrays, as assessed by light microscopy (Wood, 1976). The more mature the NC + SC culture is before NC removal, the longer these extended arrays of SCs persist. Within individual arrays the positions of SC nuclei are seen to change, suggestive of SC movement within but not away from the chain. In electron micrographs of transverse sections of these still elongated SCs following NC loss, numerous processes are seen to be clustered together or nestled next to a perinuclear area, either configuration bounded by a persisting BL (Fig. 5). In many of these SC processes the cytoplasm is decreased in density, and microtubules and intermediate filaments increase in prominence (Figs. 4, 5). Mesaxon configurations are not seen. These changes
Basal Lam iza Form (1t im,
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lead to a marked resemblance of many SC processes to neurites (Figs. 2, 5). It should be stressed that SC cultures such as those illustrated had been carefully surveyed by phase microscopy before thin sectioning to confirm the absence of neuronal somata. When neurons are reintroduced into a cultured SC population, BL formation starts anew along with ensheathment of nerve fibers by SCs. This experiment was performed by adding an explant especially prepared to contain only NCs to a culture of SCs from which neurons had been removed 7 (or 12) days earlier, when the NC + SC culture was about 2 weeks old. A sibling culture demonstrated that BL was no longer present 1 week after neuron removal. Three (or 5) weeks after recombining neurons and SCs, BL was once again evident on the SC exterior (Fig. 6). We conclude from the experiments described in this section that in the absence of neurons BL may persist but that BL formation (as assessed electron microscopically) proceeds only in the presence of both NCs and SCs. NCs cultured by themselves do not exhibit patches of BL (Bunge et al., 1980). Z’rypsin treatment. Cultures of NCs + SCs or SCs alone were treated with trypsin to remove the BL and were then maintained for periods of up to 1 month to study the reappearance of BL. It was known from earlier work (Yu and R. Bunge, 1975), that trypsin treatment is not detrimental to the health of cultured neurons and SCs. At the light microscope level, SC nuclei were seen to change from an elliptical to a round configuration and the cell as a whole began to bulge from the fascicle during the incubation with trypsin. This change in SC conformation persisted for several days despite return to normal culture medium. At the electron microscope level, the BL was found to be absent, or thinned and in the process of lifting off the SC surface in cultures fixed at the completion of the hour-long trypsin treatment period (Figs. ‘7-10). The thinned BL that was separated from the SC surface for substantial distances occasionally retained attachment to localized regions of the SC surface exhibiting subplasmalemmal density (Fig. 11). These specialized regions of the SC plasmalemma were usually devoid of BL directly after treatment. SC cytoplasm appeared
FIG. 4. Eight weeks after NC removal from a 3-week NC + SC culture. The surface of each SC process remains devoid of BL. The collagen substratum upon which the outgrowth initially developed is at the bottom of the figure. X32,000. FIG. 5. Eight weeks following NC removal from a 4-month NC + SC culture. Basal lamina is visible on areas of the SC surface. These areas sometimes exhibit subplasmalemmal dense material (b) and the collagen fibrils are associated with these regions rather than with uncoated portions of SC plasmalemma. Because of the prominence of intermediate filaments and microtubules, some of the SC processes resemble neurites which are known to be absent from this preparation. X38,000. FIG. 6. Recombined NC + SC culture. NCs were removed from a 2-week NC + SC culture and then, 2 weeks later, a new explant consisting only of NCs was added to the SC population. During the ensuing R-week period, neurites (n) grew from the explant and became ensheathed. BL (b) has reappeared on the exterior of SCs related to neurites. X 22,000.
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normal but the nuclear outline had become more irregular and the configuration of the cell was changed, particularly in relation to unmyelinated axons. SC processes had pulled away from the unmyelinated neurites, leaving the neurites in greatly enlarged cytoplasmic troughs or divested of cytoplasmic ensheathment (Figs. 7-9). Many SC extensions appeared unrelated to neurites, resembling the long “meandering” processes found in NC + SC preparations cultured in a defined medium without serum (Moya et al., 1980). The relationship of the SC to its myelinated axon remained intact along most of the internode, however, even though the BL had been removed from these SCs as well (Figs. 810). The thin fibrils in the extracellular space survived the trypsin treatment as would be expected of collagenous material (Fig. 9). At ‘7 and 16 days after treatment with trypsin, reensheathment of unmyelinated axons had begun but the usual degree of ensheathment in outgrowth areas had not yet been attained, as assessed by electron microscopy. The long thin SC processes observed directly after exposure to trypsin were no longer seen at 7 days. Enlarged lysosomes were observed within the SCs (as in Figs. 13 and 14). Residues of pale BL-like substance were found external to some of the patches of SC plasmalemma underlain with dense material (Fig. 12); these columns were interpreted to be the remnants of BL that had been loosened from relatively broad expanses of SC surface. Examples of new BL could be found in relation to a few areas of SC subplasmalemmal density (see Fig. 12) in cultures with NCs. At 16 days new BL was readily apparent in NC + SC cultures. It was most prominent on SCs related to myelinated axons (Fig. 14), in some cases present in a continuous layer. Elsewhere, on SCs related to unmyelinated fibers, only short patches of BL could be visualized, usually in relation to the SC plasmalemma with subjacent dense material but sometimes extending beyond these specialized areas. It appears that reforming BL is first found opposite subplasmalemmal densities. At 30 days after employing trypsin, reensheathment of axons had advanced considerably. More extensive BL
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coverage of SCs related to unmyelinated axons could be seen (Figs. 15, 16). In contrast, reformation of BL was not observed in cultures of SCs devoid of NCs at any subsequent interval studied (Figs. 13,17,18). This lack of BL reformation was confirmed in older culture preparations, In one experiment, NCs were removed from a series of 4-monthold NC + SC cultures and, 17 days later, the remaining SCs were treated with trypsin. Electron microscopic examination of a culture fixed directly after trypsin application showed that BL had been removed, as in other experiments. Two weeks after trypsin treatment the SC surface remained essentially devoid of BL. DISCUSSION
This study demonstrates that NCs must be present for the generation of a morphologically visible BL on the SC surface. When NCs are removed from NC + SC cultures 1 month old or less, BL disappears from the SC surface. When NCs are returned to SC populations, BL reappears on the SC exterior. The lamina may be removed from SCs by trypsin, as first illustrated by Yu and Bunge (1975). If BL is removed in this way, it reappears on SCs in the company of NCs; BL is not seen on SCs that have been divested of NCs. The latter finding is in agreement with the report by Dubois-Dalcq et al. (1981) that BL does not reappear on SCs obtained from dissociated newborn rat sciatic nerve and cultured without NCs for 2 to 3 weeks. Our conclusion that the presence of NCs is required for the generation of BL on SCs is consistent with the work of Billings-Gagliardi et al. (1974) and Armati-Gulson (1980), who found that migrating SCs lack BL but subsequently acquire it upon cessation of migration and assumption of a spindleshaped contour along a neurite. Following trypsinization of NC + SC cultures, BL reappearance is faster on SCs related to myelinated axons than on SCs ensheathing unmyelinated neurites. One explanation for this observation is as follows. The trypsin treatment disrupts the SC-unmyelinated fiber relationship and the process of reensheathment requires several weeks. The myelinated axon-SC rela-
FIGS. 7-11. Appearance of NC + SC cultures directly following trypsin treatment. FIG. 7. Unmyelinated neurites are no longer closely embraced by SC cytoplasm. Some neurites have become free of ensheathing cytoplasm, as shown at the upper right. (*). X22,000. FIG. 8. In contrast to unmyelinated neurites, the myelinated axon remains within its SC encasement. The BL (b) has been markedly loosened from the exterior of the myelinating SC illustrated here. Cross-sectioned collagen fibrils lie adjacent to the external surface of the lamina. X19,000. FIG. 9. This micrograph demonstrates the disappearance of BL from the SC surface following trypsin treatment. The disrupted ensheathment of unmyelinated neurites is also depicted here. Collagen fibrils (*) survive the trypsin treatment. ~57,000. FIG. 10. This figure illustrates thinned BL (b) in the process of lifting off the exterior of a SC related to a myelinated axon. ~57,000. FIG. 11. Occasionally BL attachment to the SC exterior is retained in an area displaying subplasmalemmal density (b). Collagen fibrils are present within the lamina that has been loosened from adjacent SC surfaces. X34,000.
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tionship, on the other hand, is not disrupted by trypsin along most of the internode. Therefore, NC-SC interaction is allowed to continue in the case of myelinated axons, in contrast to the removal of the unmyelinated neurites from contiguity with the SC. BL formation on unmyelinating SCs is slower because time is required for reensheathment and reestablishment of SC-NC proximity to allow interaction. Additional observations presented here show that well-formed BL may be maintained or persists in the absence of NCs. When NCs are removed from 3- or 4month-old NC + SC cultures, BL persists for periods of up to 8% weeks, the longest interval examined. The BL is thicker and more continuous in older cultures (Bunge et al., 1980). This finding of persisting BL agrees with that which has been obtained from chronic denervation studies in animals. Weinberg and Spencer (1978) and Payer (1979) observed, as have numerous workers before them studying Wallerian degeneration for shorter time periods, that BL survives neuronal loss for months. It seems most likely to us that the persistence of BL in these situations is due to its stability rather than continuing formation. In both the work utilizing older cultures and the illustrations obtained from the animal denervation investigations (Weinberg and Spencer, 1978; and Payer, 1979) the surviving BL is found only on the external aspect of the cluster of SC processes. The uncoated SC surfaces located in the interior are probably areas that lacked BL prior to neurite loss, areas of SC surface that were situated in apposition to axolemma, for instance. If BL were reforming in the absence of neurites, it would seem likely that patches of it would be found throughout the aggregated SC processes. Our results indicate that not only does trypsin remove the BL but it also has a marked effect on the SC-unmyelinated axon relationship, as Yu and Bunge (1975) observed earlier. The SC cytoplasm no longer embraces these axons and, in many cases, the unmyelinated fibers have been completely divested of ensheathing cytoplasm. It is conceivable that the retraction of SC processes from unmyelinated axons is a secondary effect of the loss of BL. But our observation that the axonSC apposition is well maintained along much of the myelin internode despite BL loss argues that trypsin directly affects the unmyelinated axon-SC relationship.
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(Components mediating axon-SC interaction within the internode presumably are spared due to lack of penetration of trypsin.) This interpretation also fits with the observation that the SC paranodal loops, emanating from myelin and joined to the axolemma by means of specialized contacts, become detached from the axon following trypsin (Yu and Bunge, 1975). These junctional regions appear particularly susceptible to trypsin, showing evidence of damage 15 min after initiating trypsin treatment (Yu and Bunge, 1975). It may be that the neurite or SC surface or a possible liganding substance is substantially modified by trypsin treatment. Reensheathment of unmyelinated fibers and repair of paranodal structure require several weeks. In fact, reattachment of SC cytoplasmic loops to axolemma often occurs in areas that had been less accessible to trypsin at the time of treatment (Yu and Bunge, 1975). The effect of trypsin on axon-SC relationships is in contrast to that observed using collagenase; collagenase leads to the disappearance of collagen fibrils and the partial disruption of BL but not to noticeably changed unmyelinated neurite-SC relationships. Schwann cells that have been divested of neurites tend to remain in linear arrays. In fact single SCs can be shown to be highly elongated (Bunge and Bunge, 1981). The older the NC + SC culture at the time of neuron removal, the greater is the tendency for SCs to remain confined to their original fascicles. We now know that BL is retained for at least 8% weeks after NC removal from cultures several months old. This remaining BL undoubtedly aids in the retention of SCs in fascicles. In a transverse section of such a SC fascicle, electron microscopy has shown that there are varying numbers of clusters of SCs; each cluster is surrounded by BL that encloses numerous processes and sometimes a perinuclear region of a SC as well. The cytoplasmic content of these SC processes closely resembles that of SCs bereft of axons in the animal. As Payer (1979) observed, the cytoplasm in many SC processes decreases in density and microtubules and intermediate filaments become more prominent. These changes lead to a close resemblance between SC processes and neurites in many cases (see also Ochoa (1975)). It appears that there are more SC processes within a BL bounded cluster than before neurite loss. These findings are strikingly similar to those obtained by
FIG. 12. Seven days after trypsin treatment of a NC + SC culture. An occasional residue of BL (-) may be observed in association with SC subplasmalemmal densities. A few examples of typical BL (b), interpreted to be newly formed, are visible at this time interval. The new BL appears on membrane domains exhibiting subjacent dense material. Reensheathment of neurites is not yet complete. X20,000. FIG. 13. Seven days following trypsin treatment of a SC culture. Reforming BL is not visible. Some remaining BL material is present, as shown at the *. X16,000. FIG. 14. Sixteen days after trypsin treatment of a NC + SC culture. A nearly continuous BL (b) is apparent on the exterior of the SC related to the myelinated axon. In other areas of this field, only scattered patches of BL are evident on SCs associated with unmyelinated neurites. ~18,000.
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Weinberg and Spencer (1978) and Payer (1979), who studied the fine structure of SCs in denervated isolated segments of nerve remaining in the rat. Are the numerous SC processes found within one cluster bounded by BL (as viewed in electron micrographs) from a few SCs as one would expect or does each process derive from a different SC? Some of the SC processes within a BL defined cluster may arise from branching of the SC. But because space becomes available following the disappearance of neurites, SCs may further elongate along the cluster. SC nuclei are observed in living cultures to shift in position along a fascicle of SCs. The increased prominence of microtubules and intermediate filaments may be related to this exaggerated extension of SCs divested of neurites. It is unlikely that SCs, the majority of them having been related to unmyelinated neurites, increase substantially in number within the cluster bounded by BL because, in the absence of NCs, they do not incorporate [3H]thymidine (Wood and Bunge, 1975). It is likely that SCs synthesize most of the BL constituents present in NC + SC cultures grown in the absence of fibroblasts. If this BL resembles laminae of numerous other cell types, then it contains collagen (reviewed in Bailey et al., 1979; Kefalides, 1978; Miller, 1978; and Rhodes and Miller, 1978). The SC lamina does appear to be partially disrupted by collagenase (Bunge et al., 1980). When NC + SC cultures were grown in cishydroxyproline, an agent considered to perturb collagen synthesis and secretion (Uitto et al., 1978), less BL is present (Copio and Bunge, 1980). We have obtained biochemical evidence that Type I, III, and V (and possibly IV) collagen are produced by NC + SC cultures (Bunge et al., 1980). It is now known that SCs release additional substances although they have not yet been identified. Carey and Bunge (1981) discovered that more than 20 labeled polypeptides with molecular weights ranging from 15,000 to >250,000 appear in the culture medium following the administration of [3H]leucine, [3H]glucosamine, or [3”S]methionine. When the neuronal somata are removed just prior to labeling, the presence of labeled polypeptides in the culture medium is little diminished, demonstrating that the newly synthesized material is mainly from SCs. Moreover, labeling of cultures containing only NCs results in the release of only small amounts of medium polypeptides, 40% of those released by SCs. Additional work has demonstrated that when NCs and SCs are grown in the defined medium of Bottenstein FIGS. FIGS. to both FIGS.
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and Sato (1979), the release of proteins from SCs is very markedly reduced (Carey and R. Bunge, 1981). At the same time, little BL is formed and occasional distended cisterns of rough endoplasmic reticulum are observed inin the SCs (Moya et al., 1980), both morphological dicators of reduced SC secretion. When the cultures grown in defined medium are switched to normal medium, the usual pattern of labeled polypeptides is obtained in 2 days (Carey and Bunge, 1981) and substantial amounts of BL are visible on the SC surface by 10 days (Moya et al., 1980). Surprisingly, Carey and Bunge (1981) found that NCs were not needed at the time of replacement with normal medium for the release of labeled polypeptides by SCs. In fact, they also discovered that in normal medium SCs divested of NCs for 2 weeks continued to show the normal pattern of secretion as assessed by SDS polyacrylamide gel electrophoresis. Why, then, in the presence of continuing SC secretion, does BL disappear from the SC surface as determined by electron microscopy? Does the NC manufacture small amounts of a substance necessary for the construction of BL or does the NC influence the release of additional minor components by the SC? An alternative possibility is that the surface of those SCs apposed to axons is reorganized or polarized in a manner which permits anchoring of the BL. In the absence of axons this organization breaks down and BL components, if present, cannot be anchored to the SC surface. Further work is needed to discover the mechanism by which the NC interacts with the SC to cause the formation of a morphologically visible BL. We thank Artree for secretarial aid, graphic assistance. AM 12129 (both to Health.
James for laboratory assistance, Susan Mantia and Marc Davis and Robert Freund for photoThis work was supported by Grants NS 09923 and Dr. R. P. Bunge) from the National Institutes of
REFERENCES ARMATI-GLJLSON, P. (1980). Schwann cells, basement lamina, and collagen in developing rat dorsal root ganglia in vitro. Dew. Biol. 77, 213-217. BAILEY, A. J., SHELLSWELL, G. B., and DUANCE, V. C. (1979). Identification and change of collagen types in differentiating myoblasts and developing chick muscle. Nature (London) 278, 67-69. BILLINGS-GAGLIARDI, S., WEBSTER, H. DEF., and O’CONNELL, M. F. (1974). In vivo and electron microscopic observations on Schwann cells in developing tadpole nerve fibers. Amer. J. Anat. 141, 375392. BOTTENSTEIN, J. E., and SATO, G. H. (1979). Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. Nat. Acad. Sci. USA 76, 514-517.
15-18. One month after trypsin treatment. 15, 16. NC + SC culture. As illustrated in the survey and higher magnification micrographs, BL (b) coats the exterior of SCs related myelinated and unmyelinated neurites (n). X20,000; X48.000. 17.18. SC culture. The surfaces of these SCs, cultured without NCs following the trypsin treatment, are free of BL. X20,000; ~48,000.
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BUNGE, M., JEFFREY, J., and WOOD, P. (1977). Different contributions of Schwann cells and fibroblasts to the collagenous components of peripheral nerve. J. Cell Biol. 75, 161a. BUNGE, M., WILLIAMS, A., and WOOD, P. (1979). Further evidence that neurons are required for the formation of basal lamina around Schwann cells. J. Cell BioL 83 (2, Pt. 2), 130a. BUNGE, M. B., WILLIAMS, A. K., WOOD, P. M., Urrro, J., and JEFFREY, J. J. (1980). Comparison of nerve cell and nerve cell plus Schwann cell cultures, with particular emphasis on basal lamina and collagen formation. J. Cell Biol 84, 184-202. BUNGE, R. P., and BUNGE, M. B. (1981). Cues and constraints in Schwann cell development. In “Studies in Developmental Neurobiology” (W. M. Cowan, ed.), pp. 322-353. Oxford Univ. Press, New York. CAREY, D. J., and BUNGE, R. P. (1981). Factors influencing the release of proteins by cultured Schwann cells. J. Cell Bioh, 91, 666-672. COPIO, D. S., and BUNGE, M. B. (1980). Use of a proline analog to disrupt collagen synthesis prevents normal Schwann cell differentiation. J. Cell Biol. 87, 114a. DUBOIS-DALCQ, M., RENTIER, B., BARON, A., VAN EVERCOOREN, N., and BURGE, B. W. (1981). Structure and behavior of rat primary and secondary Schwann cells in vitro. Ezp. Cell Res. 131, 283-297. KEFALIDES, N. A. (1978). Current status of chemistry and structure of basement membranes. In “Biology and Chemistry of Basement Membranes” (N. A. Kefalides, ed.), pp. 215-228. Academic Press, New York. MILLER, E. J. (1978). Comparison of basement membrane collagens with interstitial collagens. In “Biology and Chemistry of Basement
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Membranes” (N. A. Kefalides, ed.), pp. 265-278. Academic Press, New York. MOYA, F., BUNGE, M. B., and BUNGE, R. P. (1980). Schwann cells proliferate but fail to differentiate in defined medium. Proc. Nat. Acd Sci. USA 77,6902-6906. OCHOA, J. (1975). Microscopic anatomy of unmyelinated nerve fibers. In “Peripheral Neuropathy” (P. J. Dyck, P. K. Thomas, and E. H. Lambert, eds.), pp. 131-150. Saunders, Philadelphia. PAYER, A. F. (1979). An ultrastructural study of Schwann cell response to axonal degeneration. J. Camp. Neural. 183, 365-384. RHODES, R. K., and MILLER, E. J. (1978). Physiochemical characterization and molecular organization of the collagen A and B chains. Biochemistry 17,3442-3448. UITTO, J. J., UITTO, J., KAO, W. W.-Y., and PROCKOP, D. J. (1978). Procollagen polypeptides containing cis-4-hydroxy-2-proline are over glycosylated and secreted as non-helical pro--y-chains. Arch. B&hem. Biophys. 185, 214-221. WEINBERG, H. J., and SPENCER, P. S. (1978). The fate of Schwann cells isolated from axonal contact. J. Neurocytol. 7, 555-569. WILLIAMS, A. K., WOOD, P., and BUNGE, M. B. (1976). Evidence that the presence of Schwann cell basal lamina depends upon interaction with neurons. J. Cell Biol. 70, 138a. WOOD, P. M. (1976). Separation of functional Schwann cells and neurons from normal peripheral nerve tissue. Bruin Res. 115,361-375. WOOD, P. M., and BUNGE, R. P. (1975). Evidence that sensory axons are mitogenic for Schwann cells. Nature (London) 256, 662-664. Yu, R., and BUNGE, R. P. (1975). Damage and repair of the peripheral myelin sheath and node of Ranvier after treatment with trypsin. J. Cell Biol. 64, l-14.
BIOLOGY
g&449-460
(19%)
Neuron-Schwann Department
Cell Interaction
in Basal Lamina Formation
MARY BARTLETTBUNGE,'ANN K.
WILLIAMS,
of Anatomy
University
and Neurobiology,
Washington
AND PATRICK School of Medicine,
Received December 10, 1981; accepted in revised fwm
M. WOOD St. Louis, Missouri
63110
March 29, 1982
The availability of tissue culture systems that allow the growth of nerve cells, Schwann cells, and fibroblasts separately or in various combinations now makes possible investigation of the role of cell interactions in the development of the peripheral nervous system. Using these systems it was earlier found that basal lamina is formed on the Schwann cell surface in cultures of sensory ganglion cells and Schwann cells without fibroblasts. It is here reported that the presence of nerve cells is required for the generation of basal lamina on the Schwann cell plasmalemma. Utilizing nerve cell-Schwann cell preparations devoid of fibroblasts, this was found in the following ways. (1) When nerve cells are removed from 3- to 5-week-old cultures, the basal lamina disappears from Schwann cells. (2) If nerve cells are added back to such Schwann cell populations, Schwann cell basal lamina reappears. (3) Removal of nerve cells from older (3-4 months) cultures does not lead to basal lamina loss; areas presumed not to have been coated with lamina before neurite degeneration remain so, suggesting that the lamina persists but is not reformed. (4) If basal lamina is removed with trypsin, it is reformed in neuron plus Schwann cell cultures but not in Schwann cell populations alone. Thus, the formation but not the persistence of Schwann cell basal lamina requires the presence of nerve cells. INTRODUCTION
This paper is one of a series describing tissue culture studies designed to elucidate the role of cell interactions in the development of the peripheral nervous system. Methods are now available for culturing rat sensory ganglion nerve cells, (NCs),’ Schwann cells (SCs) and several types of fibroblasts either separately or in various combinations (Wood, 1976; Bunge et al, 1977). The initial paper in this series compared cultures containing NCs alone with cultures containing both NCs and SCs (Bunge et al., 1980). In spite of the lack of cellular ensheathment, cultured sensory neurons bereft of other cell types develop and maintain a dense outgrowth of nerve fiber fascicles for many months. Basal lamina and collagen fibrils are not formed. When SCs accompany the neurons, nerve fibers are ensheathed as they are in situ, with either SC cytoplasm or myelin, and basal lamina (BL) and thin collagenous fibrils are generated around the SCs. In this report we present evidence that interaction between NCs and SCs is required for BL formation on the SC. This evidence has been obtained by (1) removing NCs from NC + SC cultures, with the ensuing loss of BL from the SC surface (if the cultures are of a relatively young age); (2) reintroducing NCs into cultures containing only SCs, with the subsequent reappearance of BL; and (3) comparing regeneration of BL in cultures of NCs + SCs or SCs alone after removal i To whom all correspondence should be addressed. ‘Abbreviations used: NC, nerve cell; SC, Schwann cell; BL, basal lamina; BSS, balanced salt solution.
of the BL by means of trypsin treatment. Some of these findings have been presented in preliminary form (Williams et ab, 1976; Bunge et al., 1979). MATERIALS
AND METHODS
The culture and preparatory techniques for light and electron microscopy have been published previously (Wood, 1976; Bunge et al., 1980). In brief, 17- to 21-day fetal or newborn rat (Holtzman) dorsal root ganglia were placed on a reconstituted rat tail tendon collagen substratum and fed a complex medium containing nerve growth factor. The reader is referred to the references cited above for the regimen devised to obtain NC or NC + SC cultures free of fibroblasts by the use of the antimitotic agents, cytosine arabinoside and fluorodeoxyuridine. After the antimitotic treatment, ganglia were transplanted to allow the formation of a new outgrowth in the absence of these agents. To obtain SC preparations from NC + SC cultures, the explant containing all the neuronal somata was cut out, thereby causing the demise of all nerve fibers within a few days. Carefully prepared explants essentially containing only NCs could be subsequently transplanted into cultures consisting only of SCs. It was the outgrowth area that was generated entirely in vitro that was examined for this study. Cultures were maintained for periods of a few weeks up to several months. To study the effect of neuronal loss, SC cultures were examined 1, 2, 3, 4, 7, 8, 13, 14, 41, or 60 days after extirpation of the explant from NC + SC cultures of about 1 month of in vitro life. These
449 0012-1606/82/080449-12$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
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experiments utilized five culture series. Also, neurons were removed from 3- or 4-month-old NC + SC cultures and these cultures were maintained for an additional 2- or &week period. Treated explants composed of NCs alone were added to SC preparations that had been divested of neurons previously; these cultures were examined 3 or 5 weeks later. Trypsin treatment consisted of first rinsing the NC + SC or SC culture three times in Ca-Mg-free Hanks’ balanced salt solution (BSS), incubating in 0.25% trypsin (3X crystallized, Worthington Biochemical Corp., Freehold, N. J.) in the same fluid for 1 hr at 35°C and rinsing in Earle’s BSS before fixation or return to the usual culture medium. NC + SC or SC cultures (3 or 6% weeks or 4 months old) were studied directly after trypsin treatment or 1, 3, 7, 14, 16, or 30 days later. Three series of cultures were used for this part of the study. Cultures were fixed initially in arsenate-buffered 2% glutaraldehyde with 1% sucrose after prefatory rinsing in warmed Earle’s BSS, rinsed in BSS, further fixed in 2% 0~0~ in BSS, rinsed again in BSS, sometimes stained en bloc with uranyl acetate, dehydrated in graded ethanol and propylene oxide, and embedded in Epon-Araldite (as in Bunge et al., 1980). Suitable areas in the embedded specimen were located by phase microscopy; care was taken to sample outgrowth areas that were a similar distance from the explant, and thus comparable in maturation. These were sectioned with a diamond knife in an LKB-Huxley ultramicrotome, placed on carbon-stabilized Formvar-coated grids, stained with uranyl acetate or tannic acid (Bunge et al., 1980) and lead citrate, and examined in a Philips 300 electron microscope. RESULTS
These cultures consist of a central explant containing cell bodies of sensory neurons surrounded by a halo of neurite-SC outgrowth (free of fibroblasts) several millimeters in diameter. The morphology of the NC + SC culture outgrowth has been described previously (Bunge et al., 1980). The findings germane to this paper are that cultured SCs ensheathe nerve fibers in the normal way; one SC is related to a number of smaller unmyelinated neurites or to a single larger myelinated axon. The neu-
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rite-SC partners are enveloped by BL which thickens with increasing time in culture. The lamina coats those areas of the SC surface that interface with the extracellular milieu; there are some “intramural” SC processes as well as areas of the SC surface apposed to neurites that lack BL. Discrete regions of the SC surface membrane exhibit subplasmalemmal density which in combination with apposed BL resembles a small hemidesmosome. Scattered bundles of slender collagenous fibrils (mean diameter, 18 nm) may be observed in the extracellular space, usually in association with the lamina and situated parallel to the longitudinal axis of the fascicles (Fig. 1). Neuron removal. When the explant containing the neuronal somata is cut out of NC + SC cultures, outlying neurites degenerate rapidly; deteriorating axons are seen at 1 and 2 days but neurites are no longer identifiable at 3 days after neuron removal. In these early time intervals following explant extirpation, SC lysosomal dense bodies become enlarged and more heterogeneous in content and multivesicular bodies increase in number. Basal lamina is still visible on portions of the SC surface (Fig. 2). During these early periods before BL disappearance, the lamina usually is found on SC plasmalemmal areas lying in apposition to the extracellular fibrils which persist (as in Fig. 3); this indicates that these surface domains had bordered the extracellular space instead of interfacing with neurites or intramural SC surfaces that lack basal lamina when neurites are present. Thus, it is concluded that in the absence of neurons BL persists for a few days but no new lamina is being formed on areas that had formerly been devoid of it. Although an occasional patch may be observed, most of the BL has disappeared by 7 or 8 days following NC removal from these relatively young cultures of about 1 month in vitro age (Fig. 3). An occasional cistern of granular endoplasmic reticulum is seen to be engorged with sequestered material, although most cisterns appear normal in width and content. Basal lamina does not reappear in sibling cultures divested of NCs for 6 or 8 weeks (Fig. 4). The time course for BL disappearance from monthold cultures was consistent from experiment to experiment with one exception. In one series of cultures, ex-
FIG. 1. Control NC + SC culture. Schwann cell cytoplasm associated with neurites (n) is covered by BL (b). External to the BL collagenous fibrils that have been cross-sectioned. This and all other figures illustrate areas of transversely sectioned outgrowth formed in vitro and did not contain fibroblasts. Three weeks in vitro; x27.000. FIG. 2. Three days after NC removal from a NC + SC preparation in the same culture series as Fig. 1. Basal lamina is visible (see ,) but not all areas of the SC surface. The neurites degenerate within this time period. X38,000. FIG. 3. Seven days following NC removal from another sibling culture. The BL has vanished except for an occasional thinned (b) that is situated near collagen fibrils. Rough endoplasmic reticulum is prominent in one area of SC cytoplasm (at the micrograph ~36,000.
are thin that was on some segment center).
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amined 1, 3, 7, and 14 days after neuron removal, patches of BL were still evident in the 14-day sample. A notable difference between this culture series and the others was the presence of a greater than usual number of collagenous fibrils (see Fig. 9 in Bunge et al., 1980), suggesting that there had been greater secretory activity in this series than in the others studied. Enhanced secretory activity may have led to the formation of a more stable lamina in this culture period. This interpretation is consistent with results obtained from older cultures. When neurons are removed from 3- or 4-month-old NC + SC cultures, BL is still present 8 weeks later (Fig. 5). The lamina was undoubtedly more stable at these later times of NC removal because we have observed that it becomes more continuous and increases in thickness with time in culture (Bunge et al., 1980). At 3 or 4 weeks in culture, the lamina may be thin and discontinuous in contrast to that of older cultures. As noted after neuron removal from younger cultures, when basal laminae are present 8 weeks after neuronal loss from 3- or 4-month-old cultures, they are observed only on certain regions of the SC surface. The interpretation that these patches contain persisting rather than reforming lamina material in the absence of accompanying NCs prompted the trypsin experiments described in the next section. In spite of the lack of neurite contact for periods up to 4 weeks, SCs remain in chains of elongated cells on the collagen substratum and do not migrate from these extended arrays, as assessed by light microscopy (Wood, 1976). The more mature the NC + SC culture is before NC removal, the longer these extended arrays of SCs persist. Within individual arrays the positions of SC nuclei are seen to change, suggestive of SC movement within but not away from the chain. In electron micrographs of transverse sections of these still elongated SCs following NC loss, numerous processes are seen to be clustered together or nestled next to a perinuclear area, either configuration bounded by a persisting BL (Fig. 5). In many of these SC processes the cytoplasm is decreased in density, and microtubules and intermediate filaments increase in prominence (Figs. 4, 5). Mesaxon configurations are not seen. These changes
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lead to a marked resemblance of many SC processes to neurites (Figs. 2, 5). It should be stressed that SC cultures such as those illustrated had been carefully surveyed by phase microscopy before thin sectioning to confirm the absence of neuronal somata. When neurons are reintroduced into a cultured SC population, BL formation starts anew along with ensheathment of nerve fibers by SCs. This experiment was performed by adding an explant especially prepared to contain only NCs to a culture of SCs from which neurons had been removed 7 (or 12) days earlier, when the NC + SC culture was about 2 weeks old. A sibling culture demonstrated that BL was no longer present 1 week after neuron removal. Three (or 5) weeks after recombining neurons and SCs, BL was once again evident on the SC exterior (Fig. 6). We conclude from the experiments described in this section that in the absence of neurons BL may persist but that BL formation (as assessed electron microscopically) proceeds only in the presence of both NCs and SCs. NCs cultured by themselves do not exhibit patches of BL (Bunge et al., 1980). Z’rypsin treatment. Cultures of NCs + SCs or SCs alone were treated with trypsin to remove the BL and were then maintained for periods of up to 1 month to study the reappearance of BL. It was known from earlier work (Yu and R. Bunge, 1975), that trypsin treatment is not detrimental to the health of cultured neurons and SCs. At the light microscope level, SC nuclei were seen to change from an elliptical to a round configuration and the cell as a whole began to bulge from the fascicle during the incubation with trypsin. This change in SC conformation persisted for several days despite return to normal culture medium. At the electron microscope level, the BL was found to be absent, or thinned and in the process of lifting off the SC surface in cultures fixed at the completion of the hour-long trypsin treatment period (Figs. ‘7-10). The thinned BL that was separated from the SC surface for substantial distances occasionally retained attachment to localized regions of the SC surface exhibiting subplasmalemmal density (Fig. 11). These specialized regions of the SC plasmalemma were usually devoid of BL directly after treatment. SC cytoplasm appeared
FIG. 4. Eight weeks after NC removal from a 3-week NC + SC culture. The surface of each SC process remains devoid of BL. The collagen substratum upon which the outgrowth initially developed is at the bottom of the figure. X32,000. FIG. 5. Eight weeks following NC removal from a 4-month NC + SC culture. Basal lamina is visible on areas of the SC surface. These areas sometimes exhibit subplasmalemmal dense material (b) and the collagen fibrils are associated with these regions rather than with uncoated portions of SC plasmalemma. Because of the prominence of intermediate filaments and microtubules, some of the SC processes resemble neurites which are known to be absent from this preparation. X38,000. FIG. 6. Recombined NC + SC culture. NCs were removed from a 2-week NC + SC culture and then, 2 weeks later, a new explant consisting only of NCs was added to the SC population. During the ensuing R-week period, neurites (n) grew from the explant and became ensheathed. BL (b) has reappeared on the exterior of SCs related to neurites. X 22,000.
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normal but the nuclear outline had become more irregular and the configuration of the cell was changed, particularly in relation to unmyelinated axons. SC processes had pulled away from the unmyelinated neurites, leaving the neurites in greatly enlarged cytoplasmic troughs or divested of cytoplasmic ensheathment (Figs. 7-9). Many SC extensions appeared unrelated to neurites, resembling the long “meandering” processes found in NC + SC preparations cultured in a defined medium without serum (Moya et al., 1980). The relationship of the SC to its myelinated axon remained intact along most of the internode, however, even though the BL had been removed from these SCs as well (Figs. 810). The thin fibrils in the extracellular space survived the trypsin treatment as would be expected of collagenous material (Fig. 9). At ‘7 and 16 days after treatment with trypsin, reensheathment of unmyelinated axons had begun but the usual degree of ensheathment in outgrowth areas had not yet been attained, as assessed by electron microscopy. The long thin SC processes observed directly after exposure to trypsin were no longer seen at 7 days. Enlarged lysosomes were observed within the SCs (as in Figs. 13 and 14). Residues of pale BL-like substance were found external to some of the patches of SC plasmalemma underlain with dense material (Fig. 12); these columns were interpreted to be the remnants of BL that had been loosened from relatively broad expanses of SC surface. Examples of new BL could be found in relation to a few areas of SC subplasmalemmal density (see Fig. 12) in cultures with NCs. At 16 days new BL was readily apparent in NC + SC cultures. It was most prominent on SCs related to myelinated axons (Fig. 14), in some cases present in a continuous layer. Elsewhere, on SCs related to unmyelinated fibers, only short patches of BL could be visualized, usually in relation to the SC plasmalemma with subjacent dense material but sometimes extending beyond these specialized areas. It appears that reforming BL is first found opposite subplasmalemmal densities. At 30 days after employing trypsin, reensheathment of axons had advanced considerably. More extensive BL
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coverage of SCs related to unmyelinated axons could be seen (Figs. 15, 16). In contrast, reformation of BL was not observed in cultures of SCs devoid of NCs at any subsequent interval studied (Figs. 13,17,18). This lack of BL reformation was confirmed in older culture preparations, In one experiment, NCs were removed from a series of 4-monthold NC + SC cultures and, 17 days later, the remaining SCs were treated with trypsin. Electron microscopic examination of a culture fixed directly after trypsin application showed that BL had been removed, as in other experiments. Two weeks after trypsin treatment the SC surface remained essentially devoid of BL. DISCUSSION
This study demonstrates that NCs must be present for the generation of a morphologically visible BL on the SC surface. When NCs are removed from NC + SC cultures 1 month old or less, BL disappears from the SC surface. When NCs are returned to SC populations, BL reappears on the SC exterior. The lamina may be removed from SCs by trypsin, as first illustrated by Yu and Bunge (1975). If BL is removed in this way, it reappears on SCs in the company of NCs; BL is not seen on SCs that have been divested of NCs. The latter finding is in agreement with the report by Dubois-Dalcq et al. (1981) that BL does not reappear on SCs obtained from dissociated newborn rat sciatic nerve and cultured without NCs for 2 to 3 weeks. Our conclusion that the presence of NCs is required for the generation of BL on SCs is consistent with the work of Billings-Gagliardi et al. (1974) and Armati-Gulson (1980), who found that migrating SCs lack BL but subsequently acquire it upon cessation of migration and assumption of a spindleshaped contour along a neurite. Following trypsinization of NC + SC cultures, BL reappearance is faster on SCs related to myelinated axons than on SCs ensheathing unmyelinated neurites. One explanation for this observation is as follows. The trypsin treatment disrupts the SC-unmyelinated fiber relationship and the process of reensheathment requires several weeks. The myelinated axon-SC rela-
FIGS. 7-11. Appearance of NC + SC cultures directly following trypsin treatment. FIG. 7. Unmyelinated neurites are no longer closely embraced by SC cytoplasm. Some neurites have become free of ensheathing cytoplasm, as shown at the upper right. (*). X22,000. FIG. 8. In contrast to unmyelinated neurites, the myelinated axon remains within its SC encasement. The BL (b) has been markedly loosened from the exterior of the myelinating SC illustrated here. Cross-sectioned collagen fibrils lie adjacent to the external surface of the lamina. X19,000. FIG. 9. This micrograph demonstrates the disappearance of BL from the SC surface following trypsin treatment. The disrupted ensheathment of unmyelinated neurites is also depicted here. Collagen fibrils (*) survive the trypsin treatment. ~57,000. FIG. 10. This figure illustrates thinned BL (b) in the process of lifting off the exterior of a SC related to a myelinated axon. ~57,000. FIG. 11. Occasionally BL attachment to the SC exterior is retained in an area displaying subplasmalemmal density (b). Collagen fibrils are present within the lamina that has been loosened from adjacent SC surfaces. X34,000.
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tionship, on the other hand, is not disrupted by trypsin along most of the internode. Therefore, NC-SC interaction is allowed to continue in the case of myelinated axons, in contrast to the removal of the unmyelinated neurites from contiguity with the SC. BL formation on unmyelinating SCs is slower because time is required for reensheathment and reestablishment of SC-NC proximity to allow interaction. Additional observations presented here show that well-formed BL may be maintained or persists in the absence of NCs. When NCs are removed from 3- or 4month-old NC + SC cultures, BL persists for periods of up to 8% weeks, the longest interval examined. The BL is thicker and more continuous in older cultures (Bunge et al., 1980). This finding of persisting BL agrees with that which has been obtained from chronic denervation studies in animals. Weinberg and Spencer (1978) and Payer (1979) observed, as have numerous workers before them studying Wallerian degeneration for shorter time periods, that BL survives neuronal loss for months. It seems most likely to us that the persistence of BL in these situations is due to its stability rather than continuing formation. In both the work utilizing older cultures and the illustrations obtained from the animal denervation investigations (Weinberg and Spencer, 1978; and Payer, 1979) the surviving BL is found only on the external aspect of the cluster of SC processes. The uncoated SC surfaces located in the interior are probably areas that lacked BL prior to neurite loss, areas of SC surface that were situated in apposition to axolemma, for instance. If BL were reforming in the absence of neurites, it would seem likely that patches of it would be found throughout the aggregated SC processes. Our results indicate that not only does trypsin remove the BL but it also has a marked effect on the SC-unmyelinated axon relationship, as Yu and Bunge (1975) observed earlier. The SC cytoplasm no longer embraces these axons and, in many cases, the unmyelinated fibers have been completely divested of ensheathing cytoplasm. It is conceivable that the retraction of SC processes from unmyelinated axons is a secondary effect of the loss of BL. But our observation that the axonSC apposition is well maintained along much of the myelin internode despite BL loss argues that trypsin directly affects the unmyelinated axon-SC relationship.
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(Components mediating axon-SC interaction within the internode presumably are spared due to lack of penetration of trypsin.) This interpretation also fits with the observation that the SC paranodal loops, emanating from myelin and joined to the axolemma by means of specialized contacts, become detached from the axon following trypsin (Yu and Bunge, 1975). These junctional regions appear particularly susceptible to trypsin, showing evidence of damage 15 min after initiating trypsin treatment (Yu and Bunge, 1975). It may be that the neurite or SC surface or a possible liganding substance is substantially modified by trypsin treatment. Reensheathment of unmyelinated fibers and repair of paranodal structure require several weeks. In fact, reattachment of SC cytoplasmic loops to axolemma often occurs in areas that had been less accessible to trypsin at the time of treatment (Yu and Bunge, 1975). The effect of trypsin on axon-SC relationships is in contrast to that observed using collagenase; collagenase leads to the disappearance of collagen fibrils and the partial disruption of BL but not to noticeably changed unmyelinated neurite-SC relationships. Schwann cells that have been divested of neurites tend to remain in linear arrays. In fact single SCs can be shown to be highly elongated (Bunge and Bunge, 1981). The older the NC + SC culture at the time of neuron removal, the greater is the tendency for SCs to remain confined to their original fascicles. We now know that BL is retained for at least 8% weeks after NC removal from cultures several months old. This remaining BL undoubtedly aids in the retention of SCs in fascicles. In a transverse section of such a SC fascicle, electron microscopy has shown that there are varying numbers of clusters of SCs; each cluster is surrounded by BL that encloses numerous processes and sometimes a perinuclear region of a SC as well. The cytoplasmic content of these SC processes closely resembles that of SCs bereft of axons in the animal. As Payer (1979) observed, the cytoplasm in many SC processes decreases in density and microtubules and intermediate filaments become more prominent. These changes lead to a close resemblance between SC processes and neurites in many cases (see also Ochoa (1975)). It appears that there are more SC processes within a BL bounded cluster than before neurite loss. These findings are strikingly similar to those obtained by
FIG. 12. Seven days after trypsin treatment of a NC + SC culture. An occasional residue of BL (-) may be observed in association with SC subplasmalemmal densities. A few examples of typical BL (b), interpreted to be newly formed, are visible at this time interval. The new BL appears on membrane domains exhibiting subjacent dense material. Reensheathment of neurites is not yet complete. X20,000. FIG. 13. Seven days following trypsin treatment of a SC culture. Reforming BL is not visible. Some remaining BL material is present, as shown at the *. X16,000. FIG. 14. Sixteen days after trypsin treatment of a NC + SC culture. A nearly continuous BL (b) is apparent on the exterior of the SC related to the myelinated axon. In other areas of this field, only scattered patches of BL are evident on SCs associated with unmyelinated neurites. ~18,000.
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Weinberg and Spencer (1978) and Payer (1979), who studied the fine structure of SCs in denervated isolated segments of nerve remaining in the rat. Are the numerous SC processes found within one cluster bounded by BL (as viewed in electron micrographs) from a few SCs as one would expect or does each process derive from a different SC? Some of the SC processes within a BL defined cluster may arise from branching of the SC. But because space becomes available following the disappearance of neurites, SCs may further elongate along the cluster. SC nuclei are observed in living cultures to shift in position along a fascicle of SCs. The increased prominence of microtubules and intermediate filaments may be related to this exaggerated extension of SCs divested of neurites. It is unlikely that SCs, the majority of them having been related to unmyelinated neurites, increase substantially in number within the cluster bounded by BL because, in the absence of NCs, they do not incorporate [3H]thymidine (Wood and Bunge, 1975). It is likely that SCs synthesize most of the BL constituents present in NC + SC cultures grown in the absence of fibroblasts. If this BL resembles laminae of numerous other cell types, then it contains collagen (reviewed in Bailey et al., 1979; Kefalides, 1978; Miller, 1978; and Rhodes and Miller, 1978). The SC lamina does appear to be partially disrupted by collagenase (Bunge et al., 1980). When NC + SC cultures were grown in cishydroxyproline, an agent considered to perturb collagen synthesis and secretion (Uitto et al., 1978), less BL is present (Copio and Bunge, 1980). We have obtained biochemical evidence that Type I, III, and V (and possibly IV) collagen are produced by NC + SC cultures (Bunge et al., 1980). It is now known that SCs release additional substances although they have not yet been identified. Carey and Bunge (1981) discovered that more than 20 labeled polypeptides with molecular weights ranging from 15,000 to >250,000 appear in the culture medium following the administration of [3H]leucine, [3H]glucosamine, or [3”S]methionine. When the neuronal somata are removed just prior to labeling, the presence of labeled polypeptides in the culture medium is little diminished, demonstrating that the newly synthesized material is mainly from SCs. Moreover, labeling of cultures containing only NCs results in the release of only small amounts of medium polypeptides, 40% of those released by SCs. Additional work has demonstrated that when NCs and SCs are grown in the defined medium of Bottenstein FIGS. FIGS. to both FIGS.
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Formation
and Sato (1979), the release of proteins from SCs is very markedly reduced (Carey and R. Bunge, 1981). At the same time, little BL is formed and occasional distended cisterns of rough endoplasmic reticulum are observed inin the SCs (Moya et al., 1980), both morphological dicators of reduced SC secretion. When the cultures grown in defined medium are switched to normal medium, the usual pattern of labeled polypeptides is obtained in 2 days (Carey and Bunge, 1981) and substantial amounts of BL are visible on the SC surface by 10 days (Moya et al., 1980). Surprisingly, Carey and Bunge (1981) found that NCs were not needed at the time of replacement with normal medium for the release of labeled polypeptides by SCs. In fact, they also discovered that in normal medium SCs divested of NCs for 2 weeks continued to show the normal pattern of secretion as assessed by SDS polyacrylamide gel electrophoresis. Why, then, in the presence of continuing SC secretion, does BL disappear from the SC surface as determined by electron microscopy? Does the NC manufacture small amounts of a substance necessary for the construction of BL or does the NC influence the release of additional minor components by the SC? An alternative possibility is that the surface of those SCs apposed to axons is reorganized or polarized in a manner which permits anchoring of the BL. In the absence of axons this organization breaks down and BL components, if present, cannot be anchored to the SC surface. Further work is needed to discover the mechanism by which the NC interacts with the SC to cause the formation of a morphologically visible BL. We thank Artree for secretarial aid, graphic assistance. AM 12129 (both to Health.
James for laboratory assistance, Susan Mantia and Marc Davis and Robert Freund for photoThis work was supported by Grants NS 09923 and Dr. R. P. Bunge) from the National Institutes of
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15-18. One month after trypsin treatment. 15, 16. NC + SC culture. As illustrated in the survey and higher magnification micrographs, BL (b) coats the exterior of SCs related myelinated and unmyelinated neurites (n). X20,000; X48.000. 17.18. SC culture. The surfaces of these SCs, cultured without NCs following the trypsin treatment, are free of BL. X20,000; ~48,000.
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