Oligodendrocytes and CNS Myelin Are Nonpermissive Substrates for Neurite Growth and Fibroblast Spreading in Vi&O

The Journal of Neuroscience, July 1988, 8(7): 2381-2393

Oligodendrocytes and CNS Myelin Are Nonpermissive Substrates for Neurite Growth and Fibroblast Spreading in vi&o

Martin E. Schwab and Pica Caroni Brain Research Institute of the University of Zurich, CH-8029 Zurich, Switzerland

To study the interaction of neurons with CNS glial cells, (Tello, 1911; Ramon y Cajal, 1928; Benfey and Aguayo, 1982; dissociated sympathetic or sensory ganglion cells or fetal Richardson et al., 1984; So and Aguayo, 1985). These studies retinal cells were plated onto cultures of dissociated optic assigneda crucial role to the microenvironment of the growing nerve glial cells of young rats. Whereas astrocytes favored fibers, whereby peripheral nerve tissue should allow, support, neuron adhesion and neurite outgrowth, oligodendrocytes or provoke neurite regeneration. The involvement of neuro- differed markedly in their properties as neuronal substrates. trophic and neurotropic factors-produced by Schwanncells but Immature (O,+, A,B,+, GalCm) oligodendrocytes were fre- not by CNS glia-was suggested60 years ago by Ramon y Cajal quently contacted by neurons and neurites. In contrast, dif- (1928). In fact, a marked increase in the production of neuro- ferentiated oligodendrocytes (O,+, A$-, GalC+) represented trophic factors and cell adhesionmolecules by Schwanncells in a nonpermissive substrate for neuronal adhesion and neurite responseto denervation has recently been observed (Richardson growth. When neuroblastoma cells or 3T3 fibroblasts were and Ebendal, 1982; Longo et al., 1984; Abrahamson et al., 1986; plated into optic nerve glial cultures, the same differences Daniloff et al., 1986). However, neurotrophic factors are also were observed; differentiated oligodendrocytes were non- presentin developing and adult CNS, and increasedneurotroph- permissive for cell adhesion, neurite growth, or fibroblast ic activities were found at sites of CNS lesions(Barde et al., spreading. These nonpermissive oligodendrocytes were 1982; Korsching et al., 1985; Whittemore et al., 1985, 1986, characterized by a radial, highly branched process network, 1987; Needels et al., 1986; Shelton and Reichardt, 1986). often contained myelin basic protein, and may, therefore, Recent in vitro studies,in which dissociatedsensory or sym- correspond to cells actively involved in the production of pathetic neurons were confronted with explants of adult rat myelin-like membranes. sciatic (PNS) or optic (CNS) nerves, showedthat the differences Isolated myelin from adult rat spinal cord was adsorbed in regenerative neurite growth within peripheral or central ner- to polylysine-coated culture dishes and tested as a sub- vous tissue environments persisted in the presenceof high strate for peripheral neurons, neuroblastoma cells, or 3T3 amountsof a neurotrophic factor (NGF) (Schwaband Thoenen, cells. Again, cell attachment, neurite outgrowth, and fibro- 1985). In the same cultures, in which up to several hundred blast spreading was strongly impaired. General physico- axons could be found in the sciatic nerve explants, neurite in- chemical properties of myelin were not responsible for this growth into optic nerves was strictly absent.The samefindings effect, since myelin from rat sciatic nerves favored neuron were obtained with previously frozen optic and sciatic nerves adhesion and neurite growth as well as spreading of 3T3 that were free of living glial cells. These results strongly argue cells. These results show that differentiated oligodendro- againstthe hypothesis that the lack of CNS regenerationis main- cytes express nonpermissive substrate properties, which ly due to an absence,or insufficient production, of neurotrophic may be of importance in CNS development or regeneration. factors by denervated glial cells (Ramon y Cajal, 1928). Rather, the differentiated CNS may lack cellular or substrateconstitu- The reasonswhy the regeneration of lesioned fiber tracts is ents conducive for neurite growth during development (Liesi, almost totally absent in the CNS of higher vertebrates have 1985a; Carbonetto et al., 1987) or it may contain components remained unknown up to now. Transplantation studiesof pieces that are nonpermissive or inhibitory for nerve fiber regenera- of peripheral nerves into the adult CNS clearly demonstrated tion. the capacity of most types of CNS neurons for regenerative In the present study, dissociatedsympathetic, sensory,or ret- growth and elongation of their processesover long distances inal neurons were added to cultures of dissociated CNS glial cells grown at low cell density. One cell type with the charac- Received July 7, 1987; revised Sept. 29, 1987; accepted Nov. 6, 1987. teristics of a differentiated oligodendrocyte forming myelin We thank Ch. Milller (Munich), J. Emi, and L. Steinberg for their skillful membraneswas found to be highly nonpermissivefor neurite technical assistance. Initial experiments were done at the Max-Planck-Institute for Psychiatry, Department of Neurochemistry, Martinsried/Munich. We thank growth. This cell contact-mediated, nonpermissivesubstrate ef- Dr. H. Thoenen for his generous support and interest. The gift of antibodies by fect was observed for primary culture neurons, neuroblastoma Drs. D. Dahl (Boston), F. Omlin (Lausanne), M. Schachner (Heidelberg), and M. cells, and spreading 3T3 fibroblasts. Isolated myelin from the Willard (St. Louis) is gratefully acknowledged. We aregrateful to Mrs. S. Kaufmann for typing and to Dr. D. Kuffler for critically reading the manuscript. CNS, but not myelin from sciatic nerves, likewise inhibited This work was supported by the Swiss National Foundation for Scientific Re- neurite growth and fibroblast spreading. search (Grant 3.043-0.84) and the Bon&i-Theler Foundation (Zurich). Correspondence should be addressed to Prof. M. E. Schwab, Brain Research Materials and Methods Institute of the University of Zurich, August-Forel-Strasse 1, 8029 Zurich, Swit- zerland. Glial cell cultures. Optic nerves were dissected from I- to 12-d-old or Copyright 0 1988 Society for Neuroscience 0270-6474/88/07238 l-13$02.00/0 young adult (1 N-220 gm) Wistar rats and collected in plating medium 2382 Schwab and Caroni - Oligodendrocytes Are Nonpermissive Substrates

(air-buffered enriched L,, with 5% rat serum; Mains and Patterson, and A2B, were directed against surface antigens and were therefore 1973). The meninges and blood vessels were carefully removed under incubated on the living cultures at room temperature for 30 min at a a microscope, and the nerves were cut into small pieces. Dissociation dilution of 1:20 in PBS/sucrose/BSA. Antibodies against Thy-l were of young nerves was done twice for 25 min each in 0.25% trypsin diluted 1: 10, anti-fibronectin 1:20. The cultures were then rinsed twice, (Sigma) and 0.02% collagenase (Worthington) (Raffet al., 1979) in CMF- fixed for 10 min with 4% formalin in PBS, rinsed again, incubated for PBS (Ca’+/Ma’+-free PBS) at 37°C. Adult ootic nerves were dissociated 1 hr with the labeled secondarv antibodies (dilution 1:30-l : loo), washed, in 0.1% tryp&, 0.1% collagenase for 1 hr. at 37°C followed by 0.5% and mounted in PBS:glycerol(l: 1). For double-labeling experiments of trypsin for 10 min. After washing and dissociation by trituration with A,B, or 0, antibodies with the 0, antibody, living cultures were first a Pasteur pipet, the cells were plated into the wells of 35 mm tissue incubated with antibodies A,B, or O,, followed by anti-mouse-FITC culture dishes containing 4 internal wells at a density of 20,000-30,000 and then with antibody 0,; after fixation, this was followed by anti- cells/well (surface of well, 95 mm’). For 7- to IO-d-old optic nerves, the mouse-RITC. In order to detect A,B, or 0, labeled cells which do not yield was about 10,000 cells per nerve. The culture substrate for most carry the 0, antigen, the sequence was reversed in some experiments. of the experiments was polyomithine (PORN, Sigma, 0.5 mg/ml in Staining for GFAP was done on cultures previously fixed in 95% eth- borate buffer, incubated overnight) or polylysine (PLYS, Sigma, 50 pg/ anol/5% acetic acid for 30 min at 4°C and rehydrated into PBS. In the ml in water); in some experiments, a dried collagen film (calf skin case of O,/GFAP double-labeling experiments, staining with the 0, collagen, incubation overnight with sterile solution), laminin-coated antibody was done first on the living cultures, followed by 10 min PORN [purified mouse EHS tumor laminin (gift of Dr. R. Timpl, Mu- fixation in 4% formalin and then by ethanol/acetic acid treatment and nich), 5 fig/ml, incubated for 3 hr on dishes previously coated with GFAP staining. For visualization of MBP, the cultures were briefly fixed PORN], or plain tissue culture plastic was used. The culture medium in 4% formalin, then treated with ethanol/acetic acid, and finally in- was an enriched L, 5 medium with 5% rat serum, penicillin (100 U/ml), cubated with anti-MBP antiserum (1:500) for 1 hr at room temperature. and streptomycin (100 Ilg/ml) (Mains and Patterson, 1973). In some Ethanol/acetic acid fixation was also used for visualization of neurofila- experiments, 10% fetal calf serum (FCS) was added instead of the rat ments. serum. Evaluation of double-labeled cultures. Cultures were systematically Optic nerves of E 13 or E 17 chicken embryos were dissociated by brief screened in the fluorescence microscope for the presence of one antigen trypsin/collagenase treatment and cultured for 2-7 d in L,, with 5% (usually 0,), and every labeled cell was examined for the presence of FCS on PORN-coated culture dishes. the other antigen, e.g., AZB,, 0,, or GFAP. Glia-nerve cell cocultures. Three different types of nerve cells were Evaluation of cocultures with nerve cells, neuroblastoma cells, or 3T3 cocultured with glial cells: sympathetic neurons from the superior cer- cells. Antibody-labeled cultures were systematically screened in the flu- vical ganglion of newborn rats, sensory neurons from dorsal root ganglia orescence microscope, and all O,-labeled cells were photographed. The of newborn rats, or cells from the retina of E17-El8 embryonic rats. same fields were photographed under phase-contrast illumination. The Superior cervical and dorsal root ganglia were dissected and dissociated oligodendrocyte surface area occupied by or in contact with neurons, into single cells as described by Mains and Patterson (1973) and Schwab neurites, ganglionic Schwann cells, or 3T3 cells was estimated, and the and Thoenen (1985). Retinas were dissected from the embryos, cleaned oligodendrocytes were grouped into 3 categories: cells with ~20, 20- from adhering blood vessels, incubated in 0.03% trypsin and 0.03% 80, or > 80% of the territory covered by neurons, neurites, or 3T3 cells. DNAase for 10 min at 37°C washed by centrifugation in serum-con- (Single thin processes, especially of immature cells, were often excluded taining medium, and dissociated by trituration. from evaluation for reasons of comparability with the dense process Theneurons were added to 2- to lo-d-old glial cultures in the same network of highly branched oligodendrocytes.) In experiments with ret- medium. with the addition of NGF (2.5s NGF. 50 or 100 r&ml) for inal cells, total oligodendrocyte territory and areas overlapped by retinal sensory and sympathetic neurons or brain-derived neurotrophic factor cells were measured with a Hewlett-Packard digitizer. The oligoden- for the retinal cells (Johnson et al., 1986). In order to suppress the drocyte subtypes were identified on the corresponding fluorescence mi- Schwann cells added together with the peripheral neurons, pulses of crographs. The criteria used were cell morphology and antigenic char- cytosine arabinoside (Ara C, 10m5M) were given twice for 24 hr on the acteristics (0,/O,). A,B, staining could not be used as a marker for 2nd and 5th d of coculture in some experiments. The cultures were immature cells, since this antigen was rapidly lost (without a concom- processed for antibody staining after l-5 d of coculture in the case of itant change in cell morphology-unpublished observations) after co- retinal cells or after 2 d-3 weeks in the case of peripheral ganglion cells. culture with neurons. The distinguishing morphological criteria were Mouse neuroblastoma cells (line NB-2A) cultured in DMEM/lO% shape and size of the cell body, number of primary processes, branching FCS were detached from the culture flasks by a brief treatment with pattern of processes, and the occurrence of anastomoses and membrane 0.1% trypsin in CMF-Hank’s solution terminated by addition of DMEM/ sheets within the process network. With these criteria, highly branched FCS. After washing, the cells were added to glial cultures (40,000 or oligodendrocytes and immature oligodendrocytes could be reproducibly 20,000 cells/well) in DMEM/FCS with either 2 mM dibutyryl-cyclic distinguished. Most (but not all) of the highly branched cells were pos- AMP or glia-derived neurite-promoting factor (GdNPF) (Guenther et itive for the 0, antigen; immature cells were consistently negative. al., 1986). Quantification of the direction of neuroblastoma process outgrowth Mouse NIH 3T3 cells, treated identically to the neuroblastoma cells, with respect to highly branched oligodendrocytes was done as illustrated were added to 2- to 3-d-old cultures of 7-d-old or newborn rat optic in Figure 6. Highly branched oligodendrocytes were sampled system- nerves at a concentration of 20,000 or 40,000 cells/well in DMEM atically, and neighboring neuroblastoma cells were classified as “adja- containing 10% fetal calf serum or in MEMa supplied with insulin (20 cent” if the distance between the edge of the oligodendrocyte process @ml) and transferrin (50 &ml). Cultures were returned to the incu- network and the NB-2A cell was less than 2 cell body diameters. Further bator for 2-4 hr and then fixed with warm 4% formalin in phosphate cells were classified as “distant” (see Fig. 5). A circle with 8 sectors (4 buffer and double-stained with the 0, and 0, antibodies. classes) was overlaid over the center of each neuroblastoma cell and Immunojluorescence. The following antibodies as markers for oli- oriented towards the nearest oligodendrocyte cell body; the neuroblas- godendrocytes, astrocytes, neurons, or fibroblasts were used: oligoden- toma processes were then counted in each sector (see Fig. 6 and Table drocytes: mouse monoclonal antibody (m-AB) 0, (Sommer and Schach- 2). ner, 1981); mouse m-AB 0, (Sommer and Schachner, 1981); specific Preparation of myelin. Spinal cords were dissected from 200 gm rats, for galactocerebroside (GalC; Singh and Pfeiffer, 1985); and goat anti- carefully cleaned from adhering dorsal and ventral roots, and homog- serum against myelin basic protein of rabbits (Omlin et al., 1982); enized (Polytron, 30 set at half-maximal speed). Sciatic nerves were precursor cells: mouse m-AB A,B, (Sera-Lab, Crawley Down, GB); as- dissected, minced, and homogenized. Myelin fractions were isolated by trocytes: rabbit antiserum against glial fibrillary acid protein (GFAP) flotation of low-speed supematants on sucrose density gradients (Col- (Dahl and Bignami, 1976); neurons: mouse m-AB against guinea pig or man et al., 1982). In some experiments, to remove possible trapped rabbit neurofilaments (Willard and Simon, 198 1);jibroblasts: mouse m- contaminants, the crude membrane fraction was washed following hy- AB 0x7 against Thy-l. 1 (Sera-Lab); goat antiserum against human potonic shock. Sedimentation in hypotonic medium was achieved at fibronectin (LETS protein; Cappel, NC). 10,000 x g for 5 min. Membrane fractions in sucrose solutions con- The specific antibodies were visualized by the corresponding anti- taining no more than 50 mM ionic species were adsorbed for several mouse. anti-rabbit. or anti-aoat-FITC or -RITC linked secondarv an- hours onto the wells of polylysine-coated tissue culture dishes (about tibodies (Cappel, NC). Priorto staining, the cultures were washed iwice 0.1 mg of protein/cm* of tissue culture dish). Unbound membranes with PBS containing 5% sucrose and 0.1% BSA. The antibodies 0,, O,, were removed by 3 washes with CMF-Hank’s solution. Coated dishes The Journal of Neuroscience, July 1988, L?(7) 2383

Figure 1. Typical morphologies and antigenic characteristics of immature oligodendrocytes (a) and highly branched oligodendrocytes (b-f). The antigenic profile (A&, O,+, O,+, often MBP+) suggests that highly branched oligodendrocytes are actively involved in myelin synthesis. a and b, Double-stained culture of 7-d-old optic nerve glial cells (2 d in vitro): A,B, (a) labels immature oligodendrocytes and type II astrocytes; 0, (b) exclusively labels highly branched oligodendrocytes. x 200. c and d, Highly branched oligodendrocytes from adult rat optic nerves (8 d in vitro) stained with antibody 0, (c) or 0, (6). x 300. e, Highly branched oligodendrocyte from lo-d-old rat optic nerve (14 d in vitro) stained with 0,. Process network contains flat membrane areas. x 300. f; MBP-positive oligodendrocyte (8-d-old optic nerve, 2 d in vitro). x 700. 2384 Schwab and Caroni - Oligodendrocytes Are Nonpermissive Substrates

Table 1. Oligodendrocyte subpopulations characterized by antibody labeling

Percentage of labeled cells Population A,B,+/O,- A,B,+/O,+ A&/O,+ A,B,+/O,- A,B,+/O,+ A&/O,+ Highly branched oligodendrocytes 0 9*4 91*4 0 1+2 93 ? 2 Cells with irregular or polygonal shapes Flat, membranous cells 37 * 4 51 +6 12 t 6 100 0 0 Process-bearing cells 18 * 5 14 + 5 8?2 84 k 6 14 k 6 1.5 +z 1.5 Cells with filopodia 0 57 k 8 43 k 8 91 1 (8 k sy Dissociated 7- to 1 O-d-old rat optic nerve cells were cultured on polyornithine for 2 d and labeled by either first antibody A,B, (detected by anti-mous+FITC) followed by 0, or 0, (detected by anti-mouse+RITC) or vice versa. The proportion of double-labeled cells was calculated from the values obtained for A,B,+/O,,; and A&/ O,,,+ cells. Values represent the means f SEM of 4-6 cultures (120-200 cells/culture) from 2 separate experiments. n This population of A,B,+/O,m cells contains type II astrocytes and precursor cells not expressing any oligodendrocyte marker. h Variable, weak, granular staining. were then immediately used in substrate testing experiments. In exper- dendrocytes. Their characteristic processnetwork may be the iments with sympathetic or sensory neurons, small droplets of central result of an unstable, partially collapsedmyelin membranecon- or peripheral myelin were deposited in defined patterns over 35 mm culture dishes. taining occasionalflat membrane areas (Figs. le. 54. Sympathetic or sensory neurons cultured as described before were The total yield of cells from adult nerves was very low. Both examined after 12 hr-4 d, neuroblastoma cells after 5-24 hr, and 3T3 differentiated O,-positive highly branched oligodendrocytes (Fig. cells after l-4 hr. For quantification, neuroblastoma cells were classified 1d) and immature A,B,-positive oligodendrocytes were also as round cells, cells with filopodia or short processes, or cells with present in cultures of adult tissue. processes longer than one cell body diameter. 3T3 cells were classified as round cells, cells with filopodia or short processes, or large flat cells. Three to four micrographs per culture were taken at random from 3 Coculture with sympathetic or sensoryneurons cultures for each experimental point. Dissociated cells from newborn rat superior cervical ganglia or dorsal root ganglia were added to glial cells after 2-10 d in Results culture. In part of the experiments, ganglionic Schwann cells Cultures of dissociatedyoung or adult rat optic nerves and fibroblasts were eliminated by pulsesof Ara C. NGF (50 or GFAP-positive astrocytes accounted for about 30% of the cells 100 ng/ml)‘was added to the culture medium, leadingto a rapid in dissociated 1O-d-old rat optic nerves. About 50% of the cells fiber outgrowth and to the formation of denseneurite networks were positive for the 0, antigen, a marker for differentiated, within a few days. NGF alone had no effect on the occurrence (GalC-positive) and immature (A,B,-positive) oligodendro- and morphology of oligodendrocytes.Glial cell types were iden- cytes. No overlap wasseen in the labelingbetween 0, and GFAP tified by antibody staining at the end of the experiments (2 d- or 0, and Thy- 1, confirming the specificity of the 0, antibody 2 weeks of coculture). as a marker for the oligodendrocyte family (Sommer and In cultures with a dense neurite plexus, the most striking Schachner, 1981). Thy- 1-positive fibroblastswith large flat mor- observation was the occurrence of “windows” free of neurites, phologiesaccounted for about 20% of the cells in young rat optic in the center of which cells with radial, highly branched pro- nerves. cessescould be observed (Fig. 2). Antibody staining identified thesecells as highly branched oligodendrocytes.A quantification Subtypesof oligodendrocytes of the interaction of oligodendrocyteswith sympathetic ganglion In culturesfrom 7- to 1O-d-old rats, about 50% ofthe O,-positive cells is shown in Figure 3, A, B. Astrocytes adjacent to oligo- cells were A,B,-positive. A,B,-labeled cells were O,-negative dendrocytes were rare in these cultures since the overall glial (Table 1; Fig. 1, a, b) and had different morphologies,including cell density was low; preferential association with astrocytes, cells with irregular processesfrom polygonal cell bodies, flat therefore, could not account for this result. Highly branched cells with peripheral processes,bipolar cells, or cells decorated oligodendrocytes excluded neurons from their territory, irre- with filopodia. On the basisof this marker profile (A,B,+, O,+, spective of the culture substrate used. The same “windows” O,m), and in agreement with Schnitzer and Schachner (1982), were formed on plain plastic, collagen, polyomithine-, or lam- we interpret these cells as being precursor and immature oli- inin-coated culture dishes.No difference was seenbetween sym- godendrocytes. They are collectively called “immature oligo- pathetic and sensory neurons; both were excluded from the dendrocytes” in the following. This cell group is probably het- territory of highly branched oligodenchocytes. Likewise, Schwann erogenous, as suggestedalso by the different morphologies. cells, when present, did not invade or overgrow the oligoden- Filopodia-carrying cells may be the most advanced (Table 1). drocyte processnetworks (Fig. 2b). In contrast, immature oli- About 50% of the O,-positive cells were A,B,-negative and godendrocytes, characterized by their irregular shapesand the O,-positive after 2 d in culture under our culture conditions. absenceof 0, antigen, did allow neurite growth on their pro- Most of these cells showed a typical, highly branched radial cessesand cell bodies (Figs. 2 e, f, 3B). A,B, could not be used processnetwork. Becauseof this characteristic morphology we as a marker for immature oligodendrocytesin cocultures with called these cells highly branched oligodendrocytes(Fig. 1, b-f; neurons, as this antigen was rapidly lost after addition of the Table 1). After 2 d in culture, most highly branched oligoden- neurons(M. Schwab, unpublished observations).Recent direct drocytes from optic nerves of lo-d-old rats were stained with observations of the encounter of growth cones with oligoden- an antiserum againstmyelin basic protein (MBP) (Fig. la. We drocytes showedus that growth cone movement is arrestedafter therefore interpret these cells as being myelin-forming oligo- filopodial contact is established.Normal growth cone activity Figure 2. Sympathetic (a-f) or retinal (g, h) neurons plated into cultures of optic nerve glial cells show nonpermissive substrate effect of highly branched oligodendrocytes and its absence in immature oligodendrocytes. a and b, “Windows” formed by highly branched oligodendrocytes (1 O- d-old optic nerves, 18 d in vitro) in the neurite plexus of sympathetic neurons (a, 8 d in vitro; b, 4 d in vitro). x 120. In b, a neurite changing its direction is seen (arrowhead). Schwann cells also avoid the oligodendrocyte. c and d, O,-positive oligodendrocytes (from lo-d-old optic nerves, 23 d in vitro) surrounded by plexus of sympathetic neurites (13 d in vitro). x 220. Neurites characteristically “loop around” the oligodendrocytes. The occasional spanning of neurite bundles over nonpermissive oligodendrocytes occurs as a secondary event. a and c, no astrocytes are present in the field. e and J; O,-positive cells with typical morphology of immature oligodendrocytes are permissive for sympathetic neurites (arrowheads) (5 d in vitro). x 380. g and h, E20 rat retinal cells (2 d in vitro) do not adhere or grow neurites onto highly branched, O,-positive (h) oligodendrocyte. x 200. 2388 Schwab and Caroni * Oiigodendrocytes Are Nonpermissive Substrates

Highly branched oligodendrocytes Figure 3. A and B, Histograms showing the frequency of interactions/overlap of sympathetic neurites and A C Schwann cells with highly branched (A) or immature (B)oligodendrocytes. Glial cells from 8- to IO-d-old optic nerves (2 d in vitro) were cocultured for ad- 50 ditional 2 d with dissociated neurons from superior cervical ganglia and then stained with 0,. Oligodendrocytes were classified by morphology on coded fluorescence pictures. On phase-contrast pictures, the fractional area of contact with neurites and Schwann cells was determined and classified into 3 cate- 10 gories: ~20, 20-80, or >80% of oli- v godendrocyte territory covered by neurites or Schwann cells. Values represent mean frequencies of cells in the 3 categories k SEM (4 cultures; 70-l 30 immature oligodendrocytes systematically sampled cells per culture). Cand D, Histograms showing the interaction on retinal cells with highly D branched (C) or immature (D) oligodendrocytes. Glial cultures from adult rat optic nerves (6-l 1 d in vitro) were cocultured for l-5 d with embryonic rat retinal cells. O,-stained oligodendrocytes were classified morphologically, and the total area occupied by each oligodendrocyte, as well as the fraction oc- O-20 21-40 41-60 61-60 IL81-100 cupied by retinal cells, was determined Fraction of oligodendrocyte surface by measuring with a graphic tablet. n = in contact with sympathetic neurons % of oligodendrocyte surface in contact 109. or Schwann cells with retina cells was seen during contact and crossing of immature cells (T. behavior” towards highly branched oligodendrocytes. 3T3 cells Zachleder and M. Schwab, unpublished observations). These plated at high cell density into optic nerve glial cultures attached observations also exclude that the “windows” were formed sec- and flattened out between 30 min and 3 hr on the polyomithine ondarily in the neurite plexus. substrate. In these forming monolayers, characteristic “win- Astrocytes in the samecultures were often overgrown by single dows” appeared corresponding to the territories of highly neurites or neurite bundles (Fig. 4, a, b). This was true for both branched oligodendrocytes (Fig. 5c--. At the sites of contact morphological types, flat and stellate cells. 3T3 cells formed a crescent-shapedbulge of cytoplasm. La- mellipodia were absent in this region. Significantly, fibroblasts Cocultures with fetal rat retinal cells that landed directly on highly branched oligodendrocytescom- After plating retinal cells at monolayer density on top of 5-d- pletely failed to spread.As for neurons, immature oligodendro- old cultures of optic nerve non-neuronal cells, a typical rear- cytes were not visibly avoided by 3T3 cells (Figs. 5, e, A 7). rangement of the retinal cells was observed: whereasoligoden- drocyte precursor cells were often contacted by retinal cells, the Absence of speciesspecificity highly branchedoligodendrocytes were mostly free of them (Figs. In addition to O,-positive/A,B,-positive precursor cells, dis- 2, g, h; 3, C, D). Again, astrocytes were preferred as a substrate sociatednon-neuronal cells from E 13 and E 17 chick optic nerve over polyornithine (Fig. 4, c, d). also contained the characteristic O,-positive/A,B,-negative/O,- positive highly branched oligodendrocytes. 3T3 cells plated on Responseof other cell types to highly branched top of chicken non-neuronal cells formed characteristic “win- oligodendrocytes dows” around thesechick oligodendrocytes.These findings show Neuroblastomacells (line NB-2A) were plated at high cell den- that neither the specific morphology nor the unfavorable sub- sity into dissociatedoptic nerve cultures and stimulated for fiber strate property of oligodendrocytesis speciesspecific (data not production by 2 mM dibutyryl-cyclic AMP or by GdNPF. At shown). 7, 24, or 48 hr, the cultures were fixed and oligodendrocytes were identified by antibodies 0, and 0,. Again, the territories Myelin as a substrate of highly branched oligodendrocyteswere clearly sparedby neu- Sincemyelin consistsof spirally wrapped oligodendrocyte mem- roblastoma cells (Fig. 5, a, b). Processesproduced by neuroblas- branes, we were interested in testing the properties of myelin toma cells situated closeto oligodendrocyteswere pointing away as a substratefor neuronsor fibroblasts. Crude myelin fractions from the oligodendrocytes (Figs. 5, a, b; 6 and Table 2). from adult rat spinal cord or sciatic nerve were prepared by Primary culture jibroblasts and astrocytes in the optic nerve flotation on a sucrosegradient. The myelin was adsorbed to preparations and mouse 3T3 cells showed a drastic “avoidance polylysine-coated tissue culture dishes and tested for its sub- The Journal of Neuroscience, July 1988, B(7) 2387

Figure 4. Astrocytes represent an adhesive substrate for neurons and net&es. a and b, Sympathetic neurites (13 d in vitro) growing on reactive, GFAP-positive (b) protoplasmic astrocyte (arrow in a) (from IO-d-old rat optic nerve, 23 d in vitro). x 220. c and d, Retinal cells (from El7 retina, 2 d in vitro) adhering to astrocytes (GFAP-positive; from lo-d-old optic nerve, 9 d in vitro) with long and with short (arrow) processes. x 400.

strate properties for superior cervical ganglion cells, dorsal root sities smaller than the one of 0.85 M sucrose would not be ganglion cells, neuroblastoma cells, and 3T3 cells. All 4 cell expected to sediment. types were attaching poorly to CNS myelin and showedmarked These experiments show that, in parallel to the effects of liv- difficulty in their processoutgrowth. Sympathetic and sensory ing, highly branched oligodendrocytes, myelin from the CNS is neurons on CNS myelin remained round or produced short, also a strongly nonpermissivesubstrate for primary culture neu- abortive fibers despite the presenceof NGF (50 or 100 rig/ml; rons, neuroblastomacells, and 3T3 fibroblasts. Myelin from the Fig. 8, a, c). In contrast, long fibers were produced on islets of PNS does not show a comparable nonpermissive substrateef- sciatic nerve myelin in the same culture dishes (Fig. 8, b, d). fect. Our experiments do not exclude the possibility that PNS Small CNS myelin islets on polylysine appeared as “windows” myelin permissivenessis due to contaminating basementmem- outlined by excluded net&es, whereasPNS myelin-polylysine brane components, which would adhere tightly to the myelin boundaries were apparently not detected by growing neurites membranes. CNS myelin nonpermissiveness,however, is not (Fig. 8, c, d). due to astrocyte membranes,since a cell membranepreparation Processoutgrowth from neuroblastomacells (line NB-2A) in from CNS tissue containing minimal amounts of white matter the presenceof dibutyryl-cyclic AMP was significantly reduced (superficial cortical layers) was a permissive substratefor fibro- by CNS myelin (Fig. 9A). blast spreading(P. Caroni and M. E. Schwab, unpublished ob- Spreading of 3T3 jbroblasts was strongly inhibited by CNS servations). myelin (Figs. 9B, 10). 3T3 cells remained round or produced spindle-shapedor polygonal morphologieswith a minimal cell substrate interaction. In contrast, large flat membranes were Discussion produced within 20-30 min on polylysine and, with a somewhat Cell attachment, spreading, and motility, and, in particular, slowertime course,also on myelin from the PNS (Figs. 9B, 10). neurite outgrowth, are strongly dependent on cell-substrate in- Nonpermissivenesswas associated,at least in large part, with teractions (Sanes,1983; Carbonetto, 1984). An increasingnum- myelin membranessince sedimentation at 10,000 g for 5 min ber of substratemolecules favoring neuroblast migration or neu- under hypotonic conditions (seeMaterials and Methods) was rite outgrowth are currently being found and characterized in sufficient to pellet most nonpermissivemembranes. Under these central and peripheral nervous tissue (Cornbrooks et al., 1983; conditions, most surfacemembrane components floating to den- Edelman, 1984; Liesi, 1985b; Stallcup et al., 1985; Chiu et al., 2388 Schwab and Caroni - Oligodendrocytes Are Nonpermissive Substrates

Figure 5. a and b, Highly branched oligodendrocytes (O,-positive) are nonpermissive for attachment and fiber outgrowth of NB-2A neuroblastoma cells. NB-2A cells were cultured for 24 hr on optic nerve glial cells (6-d-old rat optic nerves, 3 d in culture) and stimulated for neurite outgrowth by GdNPF (Guenther et al., 1986). NB-2A cells adjacent to oligodendrocytes (short arrows) show asymmetric outgrowth, &rtant cells (long arrows) show random orientation of outgrowth (see Fig. 6). x260. c-f; 3T3 fibroblasts plated at high cell densities into optic nerve glial cultures show nonpermissive substrate effect of highly branched oligodendrocytes (c and e). The oligodendrocyte in c and d has large membrane areas connecting its process network. An immature oligodendrocyte (e and3 arrow; O,-positive, irregular morphology) is overgrown by spreading fibroblasts. Ten- @, d) and 12- (e, j) d-old optic nerves, 2 d in vitro; 3T3 added for 3 hr. d and J 0, staining; c and d: x 300; e and J x 250.

1986; Fischer et al., 1986; Lindner et al., 1986; Mirsky et al., of this favorable substrate could be crucial for the absenceof 1986; Carbonetto et al., 1987).The appearanceof someof these neurite regenerationin the CNS of higher vertebrates (Hopkins constituentscan be correlated with specificdevelopmental stages et al., 1985; Liesi, 1985a;Carbonetto et al., 1987).In the present and, in the PNS, also with denervation (Edelman, 1984; Liesi, study, we observed that myelin-forming oligodendrocytes and 198513;Stallcup et al., 1985; Daniloff et al., 1986; Carbonetto isolated CNS myelin exert a nonpermissive substrateeffect on et al., 1987). The absenceof one such component, laminin, in outgrowing neurites of sympathetic and sensory neurons and the differentiated mammalian CNS, in contrast to the PNS or neuroblastomacells, aswell as for the attachment of retinal cells lowervertebrate CNS, suggestedthe hypothesisthat the absence and the spreadingof fibroblasts. In cerebellarcultures, a similar The Journal of Neuroscience, July 1988, 8(7) 2389

Highly branched oligodendrocytes

Immature oligodendrocytes I l+l I

Fraction of oligodendrocyte surface in contact with 3T3 cells Figure 7. Histograms showing the overlap of 3T3 cells with highly branched (A) or immature (B) oligodendrocytes. 3T3 cells were cocultured for 34 hr on optic nerve glial cells at high cell density, and cultures were fixed and stained with 0,. Oligodendrocytes were sampled systematically, classified as highly branched or immature oligodendrocytes and their overlap with 3T3 cells determined in the 3 categories indicated. Figure 6. Orientation of neuroblastoma process outgrowth in relation Values represent means & SEM of 4 experiments (70-170 cells). to highly branched oligodendrocytes. Optic nerve glial cells (2 or 6 d in vitro) were cocultured with NB-2A cells for 24 hr in the presence of GdNPF or dibutyryl CAMP. O,-positive, highly branched oligodendro- lack of association of neurons with GalC-positive oligodendrocytes were systematically sampled and neighboring neuroblastoma cells were classified as adjacent when the distance between the edge of oli- cytes, in contrast to astrocytes, was described by Hatten et al. godendrocyte process network and neuroblastoma cell body was less (1984). than 2 cell body diameters (Fig. 5, a, b). Neuroblastoma cells at greater Several classes of cells were present in short-term cultures of distances were classified as distant. Neuroblastoma processes were as- dissociated rat optic nerves: oligodendrocytes, astrocytes (GFAP- signed to 4 sectors (14) according to their direction with regard to the closest oligodendrocyte as illustrated. Values in Table 2 represent means positive), fibroblasts (Thy- 1 -, fibronectin-positive), and several & SEM of 3 experiments (60-100 neurites from 3 cultures per experi- types of precursor cells. Within the oligodendrocyte family (O,- ment). * p < 0.05; *** p < 0.001. positive; Sommer and Schachner, 198 l), one main cell subtype was characterized by the absence of the 0, antigen (GalC) and of MBP, 2 components highly characteristic of myelin (Mirsky et al., 1980), and the presence of binding sites for the antibody A,B,. A,B, was shown to be a marker for oligodendrocyte/type Table 2. Orientation of neuroblastoma processes with regard to II astrocyte precursors, type II astrocytes, and neurons (Abney highly branched oligodendrocytes et al., 1981; Schnitzer and Schachner, 1982; Raff et al., 1983). Percentage of urocesses in each sector Therefore, we considered this cell class to represent immature oligodendrocytes, probably including precursors as those de- Adjacent neuro- Distant neuro- Sector blastoma cells blastoma cells scribed by Dubois-Dalcq (1986) and Sommer and Noble (1986). The presence of 0, distinguishes these cells from the 02A pre- 1 7 + 1.4 25 + 2.4 cursors (Raffet al., 1983). These immature cells showed irregular 2 34 + 1.2 26 + 1.2 and variable morphologies with bipolar shapes or polygonal cell 3 33 + 2.7 25 k 2.3h bodies and irregular processes, often decorated with filopodia. 4 26 + 2.3 24 k 2.7 The cell class is probably heterogenous; cell division could be “p < 0.001. observed (F. Dutly and M. E. Schwab, unpublished obser- hp < 0.05. vations). The second main oligodendrocyte subclass consisted 2390 Schwab and Caroni * Oligodendrocytes Are Nonpermissive Substrates

Figure 8. Inhibition of neuriteoutgrowth by CNSmyelin as a substrate.Sympathetic neurons (from l-d-old rat superiorcervical ganglia) cultured in presenceof 100rig/ml NGF for 26 hr on polylysine-coatedculture dish containing focal spots of CNSor PNS myelin.CNS myelin (a, c) strongly inhibitsneurite outgrowth; PNS myelin(b, d) is a permissivesubstrate. In c andd, the borderof a myelinislet on the polylysineis shown (asterisk). x15.

of A,B,-negative, GalC-positive cells possessinga radial, highly The addition of neuronsto establishedglial cultures showed branched, and anastomosingprocess network. Most of these dramatic differences in substrate properties for neuronal at- highly branched oligodendrocytes in 2-d-old cultures of 10-d- tachment and fiber outgrowth among the various types of non- old rat optic nerves were positive for MBP under our culture neuronal cells. Astrocytes, in particular the flat reactive proto- conditions. We thus interpret this frequent cell type as repre- plasmic astrocytes were adhesive and favorable for neuronal senting oligodendrocytes actively involved in the synthesisof attachment and outgrowth, in agreementwith earlier observa- myelin membranes.In the absenceof axons, thesemembranes tions (Foucaud et al., 1982; Hatten et al., 1984; Noble et al., are deposited flat on the culture substrate; as such they are 1984; Fallon, 1985). Immature oligodendrocyteswere also fre- unstable and collapseto form the characteristic, anastomosing quently contactedby neuritesor nerve cell bodies.This behavior processnetwork. This cell type has been described as “hairy could have significant physiological relevance. During devel- eyeball cell” (Sommer and Schachner, 198l), and formation of opment, oligodendrocyte precursors migrate into the already whorls of compact myelin by suchcells hasbeen observed after formed axonal bundlesand extend processesto contact a certain prolongedtimes in culture (Rome et al., 1986;Yim et al., 1986). number of axons. Theseprocesses then start to enwrapand spiral Both immature and myelin-forming oligodendrocytes were around the axons,thus forming the structurecalled myelin (Wood seenin cultures of 7- to lo-d-old or adult rat optic nerves and and Bunge, 1984). also in cultures of l-d-old rat optic nerves, newborn rat spinal In sharp contrast to astrocytes and oligodendrocyte precur- cord, and adult rat corpuscallosum (unpublished observations), sors, we found that myelin-forming oligodendrocytes display as well as in cultures of spinal cord and optic nerves of E 13 or strongly nonpermissive substrate properties for neuronal at- E 17 chick embryos. Immature cells were clearly predominant tachment and fiber outgrowth, as well as for fibroblast attach- in dissociatesfrom younger stages,but the large drop in cell ment and spreading.This effect wasstrong and pronouncedeven yield upon dissociationwith increasingage precluded any quan- on laminin-coated culture dishes,which otherwise representan titative population analysis. However, immature oligodendro- excellent substratefor neurite growth (Manthorpe et al., 1983; cytes could also be obtained consistently from adult rat white Rogerset al., 1983). This effect could not be overcome by high matter tissues,confirming earlier observations by ffrench-con- dosesof NGF in cultures of sympathetic and sensoryneurons stant and Raff (1986). or GdNPF or dibutyryl-cyclic AMP in cultures of neuroblas- The Journal of Neuroscience, July 1988, 8(7) 2391

A Neuroblastoma cells, 5 hr 60

” round with filopodia or with cells short processes processes 2 1 cell diam

6 3T3 cells, 1 hr

60 2e 50 6 40 35 30 I=tf 20 10

round with fllopodiaor flat cells short processes cells Figure9. Nonpermissivesubstrate effects of CNSmyelin but notPNS myelinfor neuriteoutgrowth from neuroblastomacells (A) andfor 3T3 cellspreading (B). A, Neuroblastomacells cultured for 5 hr in presence of 2 mM dibutyryl CAMP onpolylysine (solid bars), CNS myelin-coated polylysine(hatched bars), or PNSmyelin-coated polylysine (open bars). Cellswere classified as roundcells, filopodia or shortprocess carrying cellsor cellswith processeslonger than 1 celldiameter. Values represent means* SEM of 3 cultures(250-450 cells per culture).B, 3T3 cells culturedfor 1 hr on polylysine,CNS myelin-coated polylysine, or PNS myelin-coatedpolylysine. Cells were classified as round cells, cells with filopodiaor shortprocesses, or largeflat cells.Values represent means f SEM of 3 cultures(300-400 cells per culture). toma cells. A similar or identical nonpermissive substrateproperty was associatedwith rat CNS myelin but not with myelin from peripheral nerves. The effect was strictly contact-dependent, sincenerve cells or fibroblasts grew well and were free to move in the immediate surrounding of these oligodendrocytes or of CNS myelin islets. Mouse 3T3 cells were also inhibited by chickenoligodendrocytes, showing that this effect isnot species specific.Recent biochemical studieshave shownthat the activity is due to specific myelin and oligodendrocyte membrane proteins (Caroni and Schwab, 1988a, b). As expected from the Figure10. Spreadingof 3T3 cells(1 hr) onpolylysine coated with CNS difference from peripheral myelin, the general physicochemical myelin(a), PNS myelin (b), or on polylysinealone (c). Roundcells or properties of myelin and the myelin lipids are unrelated to this cellswith shortprocesses are predominant on the CNSmyelin. x 170. effect. The fact that mobility of neuronal growth cones,as well as of fibroblast lamellipodia, is inhibited suggestsa common tracts of the CNS (Matthews and Duncan, 1971; Looney and underlying cell biological mechanism,which is currently under Elberger, 1986). During normal development, growing axons investigation. therefore probably never encounter myelin or myelinating oli- In the rat optic nerve, the peak number of axons is reached godendrocytes within their fasciclesbut, rather, interact with at embryonic day 20, followed by a dramatic loss of axons precursorsand immature oligodendrocytes.The extremely slow (Crespoet al., 1985). Oligodendrocyte precursorsappear from time courseobserved for in vitro myelination (Wood et al., 1980; El 7 on (Raff et al., 1985)and expressGalC around birth (Miller Wood and Williams, 1984)could be consistentwith a situation et al., 1985).The first myelin detectableby electron microscopy in which undifferentiated oligodendrocytes first interact with appearsat postnatal day 6 (Hildebrand and Waxman, 1984). axons and are then induced to differentiate and form myelin. This clear-cut temporal dissociationbetween axonal growth and The physiological significance of the potent nonpermissive myelin formation is also present in chicken optic nerves (Rager, substrateproperty of oligodendrocytes and myelin remains to 1980) and, although lesswell studied, in many white matter be investigated.In contrast to development,axonal growth cones 2392 Schwab and Caroni * Oligodendrocytes Are Nonpermissive Substrates or sprouts do encounter mature oligodendrocytes and myelin Bunge (1983) In vivo and in vitro observations on laminin produc- during CNS regeneration. Substrate properties of CNS tissue, tion bv Schwann cells. Proc. Natl. Acad. Sci. USA 80: 3850-3854. Crespo, b., D. D. M. O’Leary, and W. M. Cowan (1985) Changes in in particular the absence of potent neurite promoting substrates the numbers of optic nerve fibers during late prenatal and postnatal like laminin in the differentiated CNS of higher vertebrates, are development in the albino rat. Dev. 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