Does oligodendrocyte survival depend on axons?

Does oligodendrocyte survival depend on axons?

B.A. Barres*, M.D. Jacobson*, IR. Schmidt, M. Sendtnerf and M.C. Raff* ‘Medical R?warch Council Developmental Neurobiology Programme, Department of Biology, Medawar Building, University College,

London WCIE 6BT, UK and tMax-Planck-Institute for Psychiatry, Department of Neurochemistry, 8033 Planegg-Martinsried, Germany.

Background: We have shown previously that oiigodendrocytes and their precursors require signals from other cells in order to survive In culture. In addition, we have shown that about 50 % of the oiigodendrocytes produced in the developing rat optic nerve norrnaiiy die, ap- parently in a competition for the limiting amounts of survival factors. We have hypothesized that axons may control the levels of such oiigodendrocyte survival factors and that the competition-dependent death of oiigodendrocytes serves to match their numbers to the number of axons that they myeiinate. Here we test one predic- tion of this hypothesis - that the survival of developing oligodendrocytes depends on axons. Results: We show that oiigodendrocyte death occurs selectively in transected nerves in which the axons

degenerate. This ceil death is prevented by the deiiv- cry of exogenous ciiiary neurotrophic factor (CNTF) or insulin-like growth factor I (IGF-I), both of which have been shown to promote oiigodendrocyte survival in vitro. We also show that purified neurons promote the survival of purified oligodendrocytes in vitro. Conclusion: These results strongly suggest that oiigodendrocyte survival depends upon the presence of axons; they also support the hypothesis that a competition for axon-dependent survival signals normally helps adjust the number of oiigodendrocytes to the number of axons that require myeiination. The identities of these signals remain to be determined.

Current Biology 1993, 3:489-497

Background We have recently shown that the developing oligodendrocytes in the rat optic nerve behave like many developing vertebrate neurons, in that they depend on signals from other cells to survive in culture and about 50% of them die during normal development [ 11. Moreover, normal oligodendrocyte death, like normal neuronal death, can be prevented by the delivery of exogenous survival factors [ 11. These findings suggest that developing oligodendrocytes, like many developing neurons, may normally compete for limiting amounts of survival factors and that this competition might be an important mechanism for controlling their number. The only known function of oligodendrocytes is to myelinate axons in the vertebrate central nervous system. It would, therefore, make sense for axons to play an important role in regulating oligodendrocyte survival during the development of the central nervous system (CNS). We have found previously that the number of oligodendrocytes and their precursors decreases in transected neonatal optic nerves, thus indicating that axons normally influence oligodendrocyte development [ 21. These findings were consistent with the possibility that axons might promote oligodendrocyte survival. But because the optic nerves were transected before the appearance of oligodendrocytes in the nerve, the results did not exclude the equally likely possibilities that axons are required for oligodendrocyte precursor cell proliferation [3] or for the migration of oligodendrocyte precursor cells into the optic nerve 141. -. Here we provide strong evidence that axons are required for oligodehdrocyte survival. When the optic

nerve is cut after many oligodendrocytes have ap- peared, the observed number of dead oligodendrocytes increases and the number of live oligodendrocytes decreases dramatically. This cell death is prevented by the delivery of exogenous oligodendrocyte survival factors, and it does not occur in optic nerves from mutant mice, whose axons do not degenerate after transection. These results suggest that a competition for an axon-dependent survival signal may help to match the number of oligodendrocytes with the number of axons that require myelination, just as a competition for target-cell derived neurotrophic factors is thought to help match the number of developing presyrraptic neurons to the number of postsynaptic cells that require innervation [ 51.

Results Effect of transection on cell death in the developing rat optic nerve To test whether axons influence the survival of developing glial cells, we transected the right optic nerve be- hind the eye in rats on postnatal day 12 (~12), inducing the axons in the nerve to degenerate. At various times thereafter, we perfused the rats with paraformaldehyde under anaesthesia, cut longitudinal frozen sections ‘of both optic nerves, and stained them with the fluorescent nuclear dye propidium iodide. Dead (pyknotic) cells were identified, using phase-contrast microscopy, by their shrunken phase-dark appearance, or, with fluorescence microscopy, by their condensed and often fragmented nucleus, which stained intensely with propidium iodide [ 11. As shown in Figure 1, the number of pyknotic cells rapidly increased following

Correspondence to: B.A. Barres.

@ Current Biology 1993, Vol 3 No 8

490 Current Biology Vol 3 No 8

70 r T

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Days after transection

17 Control q Transected

Fig. 1. The effect of nerve transection on glial cell death in the developing optic nerve in postnatal rats. The number of dead cells per propidium iodide-stained longitudinal section of optic nerve at 3, 7 and 21 days after transection at PI2 is compared to that in the uncut control nerve. In this and the following figures, the results obtained from four animals are shown as means & S.E.M.

transection and stayed elevated thereafter for at least two weeks. Staining with monoclonal anti-neurolilament antibodies, either RT-97 [6] or SML32 [7], con- firmed that the axons had degenerated within a few days of transection (data not shown).

To test whether axons also influence the survival of mature glial cells, we transected the right optic nerve in older rats, at P50. Ceil death increased, but the increase was not observed until 14 days after tra.nsect.ion, corresponding to the time at which axonal neurolilament staining was lost: the average number of dead cells per section increased from 0.7 Ifi: 0.2 (mean f S.E.M.; n = 3) in control nerves to 17 + 1.5 at 14 days after transection, and was still elevated (16 f 0.9) after a further 14 days.

Identity of dead cells in transected optic nerves To identify the type of cells that died in transected optic nerves, frozen sections of nerves, which were transected at P12 and studied four days later, were double-labelled with propidium iodide and with antibodies raised either to glial fibrillary acidic protein (GFAP) or to SlOO to identify astrocytes, or with the RIP monoclonal antibody to identify oligodendrocytes [ 81. Although all of the pyknotic cells were GE;APnega- tive and SlOO-negative, 63 f 9% (mean f S.E.M.; n = 3) were RIP-positive, suggesting that most of the dead cells were oligodendrocytes (Fig. 2). The proportion of dead cells that were identified as oligodendrocytes is likely to be an underestimate: we have shown previously that dead oligodendrocytes rapidly lose their RIP antigen, so that only a minority of the dead oligodendrocytes in the normal developing optic nerve are RIP-positive [ 11.

Fig. 2. A confocal fluorescence micrograph of a dead oligodendrocyte in a transected optic nerve. A frozen section of optic nerve from a rat at P12, which had been transected four days previously, was stained with the RIP monoclonal antibody to detect oligodendrocytes. The antibody was detected with a FITC-conju- gated anti-mouse Ig antibody (green), and the DNA was stained with propidium iodide (red). A dead oligodendrocyte (arrow) is recognized by its pyknotic nucleus. It was possible to confirm that the dead cell was RIP-positive by examining it in successive 1 pm increments in the confocal microscope. Most of the live cells in this field are also RIP-positive; a cell that is clearly not labelled is seen at the center of the top edge of the micrograph. Bar, 5pm.

To determine the effect of optic nerve transection on glial cell numbers, we transected the optic nerve in rats at P8 and, 10 days later, measured the total amount of DNA in the nerves and the proportion of RIP-positive and SlOO-positive cells in the immunostained frozen sections of the nerves. As shown in Figure 3, the number of oligodendrocytes per nerve fell by almost 90 % over 10 days, whereas the number of astrocytes re- mained unchanged (data not shown). The total number of cells in the cut nerves decreased by 22 % (about 26000 cells) over this period, and this could be ac- counted for by the decrease in the number of oligodendrocytes. In uncut nerves, by contrast, the total number of cells, including both oligodendrocytes and astrocytes, increased more than two-fold during this

Does oligodendrocyte survival depend on axons? Barres et al. RESEARCH PAPER

period [I]. Thus, after optic nerve transection, there is a selective loss of most oligodendrocytes and no new oligodendrocytes appear to be generated.

P8 control

PI8 control

PI8 cut at P8

Fig. 3. The effect of nerve transection on the number of oligodendrocytes in postnatal optic nerve. The numbers of live oligodendrocytes per nerve from uncut P8, uncut P18, or PI8 nerve cut at P8, were determined by multiplying the percentage of live oligodendrocytes in the tissue sections (determined by immunostaining with the RIP monoclonal antibody) by the total number of cells per nerve (determined by DNA measurement).

Effect of transection on cell death in C56Bl/6-WLD mice Although the death of oligodendrocytes after optic nerve transection is consistent with the possibility that these cells depend on axons for their survival, it is also possible that the oligodendrocytes die because the vasculature to the nerve is damaged, or because the penetration ‘of survival factors into the cut nerve is impaired. To help distinguish between these possibilities, we examined the effect of optic nerve transection in wild-type C57Bl/6 and in mutant C56Bl/6- WID mice. c57~V6-w~~ mice have a genetic defect that prolongs the survival of the distal stumps of transected axons and thereby prevents the normal influx of monocytes into transected nerves in both the central and peripheral nervous systems [9].

As expected, in wild-type C57B1/6 nerves four days after a transection at P12, the number of dead cells increased five-fold compared to non-transected nerves (Fig. 4) and most of the axons had entirely degenerated, as shown by immunostaining with the RT-97 anti- neurofilament antibody (data not shown). In C57BV6- WLD mutant mice, by contrast, the number of dead cells in transected optic nerves did not increase compared to the number in non-transected nerves (Fig. 4) and, as expected, RT-97 staining indicated that the ax ons had not degenerated. Non-transected optic nerves contained the same”number of dead cells in both types of mice (Fig. 4). These 8results suggest that the increase in oligodendrocyte d&h in transected optic nerves in normal rats and mice is not because of an impairment in either blood flow or the delivery of survival factors,

and that axons do not need to be attached to their cell body in order to maintain oligodendrocyte survival.

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Fig. 4. The effect of nerve transection on cell death in the developing optic nerve in wild-type C57Bl/6 and mutant C57B1/6-WLD mice. The number of dead cells per propidium iodide-stained section from transected and control nerves from mice at PI2 is shown four days after transection.

Effect of exogenous CNTF or ICF-1 delivery on cell death after transection As an increase in macrophages is a feature of transected nerves in normal rats and mice but not in ~57~1/6- WLD mutant mice, it seemed possible that the increase in oligodendrocyte death in transected optic nerves is caused by the increased number of macrophages, rather than by the withdrawal of an axon-dependent survival factor. To help distinguish between these two possibilities, we examined the effect of delivering exogenous CNTF or IGF-1 into transected optic nerves, as we and others [ 10,111 have recently shown that CNTF promotes the survival of oligodendrocytes both in vitro and in viva and that IGF-1 promotes their survival in vitro [l] If oligodendrocytes die in cut nerves because they are deprived of an axon-dependent survival signal, delivery of exogenous survival factors should save them, whereas if they are killed by macrophages, delivery of survival factors might not be expected to save them. To deliver CNTF to the optic nerve, we injected into the subarachnoid space of P7 rats cells of the human kidney 293 cell line that were stably transfected with a cDNA encoding a secreted form of CNTF (see Materials and methods). We showed previously [l] that factors secreted by cells injected in this way are de- livered into the postnatal optic nerve within two days. Before implanting the transfected 293 cells, we showed that culture medium conditioned by them had survival activity but not mitogenic activity for purified optic nerve oligodendrocytes in vitro, as assessed by the MTT assay (see Materials and methods; 11,121) and

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by bromodeoxyuridine (BrdU) incorporation, respec- tively, whereas medium conditioned by non-transfected 293 cells had neither activity (data not shown). At PlO, three days after implanting the 293 cells, we cut the right optic nerve; the animals were sacrificed four days later (at H-4). As shown in Figure 5, the average number of dead cells per section of cut optic nerve ‘was decreased by about 80 % in animals that received CNTF- secreting 293 cells compared to those receiving non- transfected cells or no cells. The number of dead cells in the uncut nerves of these animals was also decreased by about 85 % (data not shown), as expected fr’om our previous studies [lo].

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Fig. 5. The effect on cell death of delivering exogenous CNTF into transected optic nerves. Human kidney 293 cells, which had been stably transfected with a plasmid expression vector containing a cDNA encoding rat CNTF with a mouse NGF signal sequence (see Materials and methods), were injected into the subarachnoid space of P7 rats. The right optic nerve was transected at PIO, and four days later the animals were perfused and the optic nerves were sectioned and stained with propidium iodide as described in the legend to Figure 1. The number of dead cells per optic nerve section was determined in animals that received either no cells (control), non-transfected 293 cells or CNTF-secreting 293 cells.

To deliver IGF-1 into the optic nerve, we implanted monkey COS cells that were transiently transfected with a cDNA encoding a synthetic analogue of IGF-1 that does not bind to IGF-binding proteins (see Materi- als and methods; [13]). As described above, before implanting the transfected COS cells, we showed that culture medium conditioned by them had survival activity but not mitogenic activity for the purified optic nerve oligodendrocytes in vitro, whereas medium conditioned by non-transfected COS cells had neither activity (data not shown). One day after implanting the COS cells into rats at ~7, we cut the right optic nerve, and the animals were sacrificed four days later. As shown in Fig- ure Ga, the average number of dead cells per section of cut optic nerve was decreased by about 70 % in animals that received IGF-l-secreting cells compared to those receiving non-transfected cells or no cells. The number of dead cells in uncut nerves was also decreased by about 80 % compared to controls (Fig. bb), whereas the number of mitotic cells in uncut nerves was not significantly affected (data not shown).

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Control Mock IGF-1

(b) 20

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Fig. 6. The effect on cell death of delivering exogenous ICF-1 into transected (a) and non-transected (b) optic nerve. COS-7 cells, which had been transiently transfected with a plasmid expression vector containing a cDNA encoding a synthetic analogue of IGF-1 (see Materials and methods), were injected into the subarachnoid space of (a) P7 or (b) PI3 rats. The right optic nerve was transected at (a) PS,, or (b) not transected and four days later the animals were perfused and the optic nerves were sectioned and stained with propidium iodide. The number of dead cells per optic’nerve section was determined in animals that received either no cells (co;stroI), mock transfected COS-7 cells, or ICF-l-secreting COS-7

Immunofluorescence labelling with the RT-97 anti-neu- rohlament antibody indicated that most of the axons had degenerated in transected nerves in the control, CNTF- and IGF-l-treated animals, suggesting that CNTF and IGF-1 treatment did not prevent the macrophage response in cut nerves. Taken together, these results suggest that the oligodendrocyte death in transected optic nerves is caused by the loss of an axon- dependent survival signal rather than by a cytotoxic effect of macrophages.

Effect of purified neurons on oligodendrocyte survival in vitro We previously found that both platelet-derived growth factor (PDGF) and IGF-1 promote the survival of purified, newly formed oligodendrocytes and their precursors in culture [l]. As oligodendrocytes differentiate, however, they lose their PDGF receptors 114,151 and can no longer be saved by PDGF, although they

”

Control Mock IGF-1

Does oligodendrocyte survival depend on axons? Barres et al. RESEARCH PAPER 4%

can still be saved by IGF-1 [l]. IGF-1, however, only promotes the survival of purified oligodendrocytes in culture for about two or three days, after which most of the cells die [lo], indicating that oligodendrocytes require additional signals for long-term survival. To test whet& neurons can provide the additional signals, we cultured purified oligodendrocytes with or without purified dorsal root ganglion (DRG) neurons (see Materials and methods) for six days and found that the survival of oligodendrocytes was increased 11 + 2 (mean f S.E.M.; n= 4) times by the presence of neurons.

Discussion Neurons promote oligodendrocyte development in at least two ways

Although oligodendrocyte precursor cells can dilferen- tiate into oligodendrocytes in culture in the absence of neurons [ 161, there is evidence that neurons are required for the normal development of oligodendrocytes. Few oligodendrocytes, for example, develop in neonatal transected optic nerves [2,17]. Neurons promote oligodendrocyte development in at least two ways. First, they promote the proliferation of oligodendrocyte precursor cells both in vitro [18-241 and in vivo [3]. Second, as we show here, they also promote the survival of oligodendrocytes, both in vitro and in vim

We showed previously that the ability of retinal ganglion cells to stimulate oligodendrocyte precursor cell proliferation in the developing optic nerve depends on the electrical activity in their axons: when the axons are electrically silenced by an intraocular injection of tetrodotoxin (‘ITX), the proliferation of precursor cells in the nerve stops [3], The ability of retinal ganglion cells to stimulate oligodendrocyte survival in the optic nerve, by contrast, does not depend on the electrical activity in the axons: TTX does not increase oligodendrocyte death in the nerve (BA Barres and MC Ralf, unpublished observations), and our present findings show that the distal stumps of transected axons in the cut optic nerves of c~~BI/G-wID mice (in which the degeneration of cut axons is greatly delayed 191) promote oligodendrocyte survival in viva, as do DRG neurons in vitro, even though these cells have little spontaneous electrical activity in culture [ 251.

Exogenous survival factors rescue axon-deprived oligodendrocytes The axon-dependent signalling molecules that promote the survival of oligodendrocytes and the proliferation of their precursor cells are not known. A number of known growth factors, however, have been shown to have such activities in vitro. PDGF [26,271, fibrob- last growth factor 1283, and neurotrophin3 [lo], for example, stimulate oligodendrocyte precursor cells to synthesize DNA, whereas IGF-1 [ 11, CNTF [lO,ll], and

NT-3 [lo] promote the survival of both oligodendrocytes and their precursors. PDGF promotes the survival of precursor cells and newly formed oligodendrocytes, but not of more mature oligodendrocytes [l], which no longer express PDGF receptors [14,15]: Some of these factors have also been shown to have similar effects in vivo. The delivery of exogenous PDGF [ 11 or CNTF [lo] suppresses the normal death of newly formed oligodendrocytes in the developing optic nerve, and the delivery of PDGF reverses the inhibition of precursor cell proliferation in the optic nerve caused by ‘ITX treatment [3]. In the present study, we show that the delivery of exogenous IGF-1 also suppresses the normal death of newly formed oligodendrocytes in the developing optic nerve. More importantly, we show that the delivery of IGF-1 or CNTF rescues axon-deprived oligodendrocytes in transected optic nerves, suggesting that axon-deprived oligodendrocytes die because they are deprived of survival signals. Thus, it seems that mature oligodendrocytes, like .newly formed ones, depend on signals from other cells for their survival and can be rescued by either IGF-1 or CNTF in vivo. As expected from results in vitro, oligodendrocytes in transected nerves cannot be saved by exogenous PDGF (our own unpublished observations).

How do neurons promote oligodendrocyte survival? Oligodendrocytes that die when they are deprived of survival factors in vitro, as well as those that die in vivo during normal development and after optic nerve transection, all have the morphology typical of apoptosis [ 291, suggesting that the cells die by programmed cell death. This, taken together with the observation that CNTF and IGF-1 can prevent most of the cell deaths that occur following optic nerve transection, suggests that neurons promote the survival of oligodendrocytes by suppressing an intrinsic suicide program [30] that would otherwise kill these cells by default. It is not clear whether neurons promote oligodendrocyte survival either directly or indirectly - by inducing astrocytes or other neighboring cell types to provide such signals - or whether survival promotion is the result of both mechanisms operating. Our finding that purified DRG neurons promote the shoa-term survival of purified oligodendrocytes in the absence of astrocytes or other cell types indicates that neurons can provide such signals directly, at least in culture. These neuronal survival signals may be bound to the neuronal cell surface or to the extracellular matrix, as continuous conditioning of culture medium with a high density of purified DRG neurons does not promote oligodendrocyte survival (our own unpublished observations) whereas co-culturing DRG neurons with oligodendrocytes does promote survival. Neurons may also indirectly regulate the production or the release of oligodendrocyte survival factors from astrocytes, which are known to make both CNTF and IGF-1 [31,32]. Because the long-term survival

494 Current Biology 1993, Vol 3 No 8

of oligodendrocytes in vitro depends on multiple signalling molecules [ 101, neurons need only regulate one such molecule to account for the death of axon-deprived oligodendrocytes in vivo.

The potential role of CNTF as a survival factor is es- pecially intriguing, as it lacks a signal sequence that would enable it to be secreted by the conventional ex- ocytotic secretion pathway. It has been suggested that CNTF may function as a ‘lesion factor’ that is released in response to injury [3I]. In this regard, it is interest- ing that, although CNTF immunoreactivity in astrocytes greatly increases after optic nerve transection (our own unpublished observations), oligodendrocytes still die in cut nerves, suggesting that CNTF is not released from astrocytes in significant amounts following transection. It remains possible, however, that axons promote the release of CNTF from astrocytes.

Conclusion A competition for axon-dependent survival signals may help to match oligodendrocyte and axon numbers. Our

previous finding that normal oligodendrocyte death can be inhibited by delivering exogenous PDGF suggested that, normally, developing oligodendroctyes may compete for limiting amounts of survival factors [l]. The finding that the majority of oligodendrocytes that die do so .within 2-3 days of being born suggested that cells thalt survive for 2-3 days after their final division seem to be no longer at risk. These findings, taken together with our present demonstration that oligodendrocytes depend on axons for survival, suggest a possible model for oligodendrocyte survival control. Once an oligodendrocyte precursor cell stops dividing and begins to differentiate into an oligodendrocyte, its specific re- quirements for survival signals change: it rapidly loses its PDGF receptors 114,151, for example, so that PDGF can no longer promote its survival [l] . Thus, it has only 2-3 days to contact a non-myelinated region of axon, which provides a new signal that is required for continued oligodendrocyte survival. A cell that fails to find an axon will kill itself (Fig. 7). Forcing newly formed oligodendrocytes to compete for a limiting amount of an axon-dependent survival signal would help to en- sure that the final number of oligodendrocytes is pre- cisely matched to the number (and length) of axons

Fig. 7. A tentative model for how axon-derived survival signals may help to match the number of oligodendrocytes with the number of axons. Once an oligodendrocyte precursor cell stops dividing and begins to differentiate into an oligodendrocyte, it has 2-3 days to contact a non-myelinatekf region of axon, which provides a new signal that the cell requires for continued survival. About half of the oligodendrocytes normally fail to contact an axon and consequently die.

Does oligodendrocyte survival depend on axons? Barres et al. RESEARCH PAPER 4%

requiring myelination, just as a competition for target- cell-derived neurotrophic factors is thought to l-&en- sure that the number of developing presynaptk neurons is matched to the number of postsynaptic c& requiring innervation [ 51,

Materials and methods Animals

Sprague/Dawky (S/D) rats were obtained from the breeding colony of University College London.

Preparation of newly formed oligodendrocytes Oligodendrocyte precursor cells were purified from postnatal rat optic nerves to > 99.9 % homogeneity by sequential im munopanning, as described previously [l]. Briefly, an optic nerve cell suspension was prepared enzymatically with pa- pain and the cells were passed sequentially over a series of Petri dishes coated with the anti-RAN-2 antibody (IgG; [33]), the 01 anti-GC antibody (IgM; [34]), and the A2B5 antibody (IgM; [35]). The purified oligodendrocyte precursor cells were removed from the A2B5 dish with trypsin. To prepare purified newly formed oligodendrocytes, the oligodendrocyte precursor cells were cultured in a 6Omm Fal- con tissue culture dish in serum-free Dulbecco’s Modified Ea- gle’s medium (DMEM) containing sodium pyruvate (1 mM), insulin (5 pg/ml), transferrin (100 pg/ml), bovine serum albumin (100 l.@ml), progesterone (60 rig/ml), putrescine (16 &ml), sodium selenite (4Ong/ml), thryoxine (4Ong/ml), and tri- iodothyronine (3Ong/ml). After 24 hours, the cells, which had all differentiated into oligodendrocytes, were removed by trypsinization.

Purification of dorsal root ganglion neurons DRG neurons were purified from P7 rats by an immunopanning procedure that was very similar to procedures described previously for the purification of retinal ganglion cells and oligodendrocytes [ 1,361. DRG neurons were used, rather than retinal ganglion cells, because their survival in vitro is supported by NGF, which does not support the survival of the oligodendrocytes [lo]. The DRGs wel’e dissociated in trypsin and collage- nase, as previously described [23,37], and then passed over sev- eral antibody-coated dishes to deplete the RAN-2-positive [33] and 04.positive [ 341 glial cells. Finally, the cells were incubated on an anti-Thy-l.l-antibody-coated dish (TllD7; [38]), which selected the DRG neurons [39,40]. The cells were removed from the linal dish with trypsin, as previously described [l] , and plated onto coverslips. The resulting DRG neurons were > 95 % pure, as demonstrated by neuroflament staining with the SMI- 32 monoclonal anti-neurolilament antibody [7], and by staining with rabbit antisera against SlOO (Sigma) or vimentin [41] to detect contaminating Schwann cells and fibroblasts, respec- tively. To minimize the number of contaminating cells, cultures were treated for two days after plating with cytosine arabinoside (5 PM). A detailed protocol is available upon request.

Co-culture of new/y formed oligodendrocytes and DRC neurons Approximately 10000 newly formed oligodendrocytes (see above) were plated onto each PDL- and laminin- coated 13 mm glass coverslip in a 24.well Falcon culture dish in 500~1 of serum-free medium containing insulin, as described above. On some of these coverslips, about 5000 purilied DRG neurons (see above) were also added. NGF (100 r&ml; 2.5S, Boehringer- Mannheim) was added to’& wells. After six days of culture, the number of live oligodendrocytes per field was determined by the M’IT assay [1,12]. Live cells were distinguished from the dead cells by their ability to metabolize the M?T into an insoluble

dark blue reaction product [ 121. The result given is the mean f S.E.M. of three cultures.

Quantitation of DNA in optic nerves . The amount of DNA in optic nerves was measured as described previously [l]. Briefly, optic nerves were digested in a buffer containing TES (Tris HCl 10 mM, EDTA 50 mM, SDS 0.1%) and proteinase K (200 pg/m.l> at 55°C for 36 h. The amounts of DNA in the optic nerve digests were measured using a flilorimetric method [42,43], which is based on the enhancement of flue-

rescence seen when the dye bisbenzimidazole (Hoechst 33258) binds to DNA. The DNA values were converted to cell number by dividing by 6.6 pg of DNA per cell 1441.

Propidium iodide labelling and preparation of cryostat sections of optic nerve Postnatal rats were anaesthetized with ether and perfused with 4 % paraformaldehyde. The optic nerves were incubated in 4 % paraformaldehyde at 4°C overnight and transferred to 30 % su- crose in phosphate buffered saline (PBS) until equilibrated. The nerves were frozen in O.C.T. compound and cut into 8 pm longitudinal sections with a Bright cvostat. The sections were col- lected onto gelatinized glass microscope slides, air dried, post- fixed in 70 % ethanol for 10 minutes at - 2O”C, and stained with propidium iodide (4 pg’ml) solution in MEM/Hepes containing DNAse-free RNAse A (100 p&l) for 30 minutes at 37°C. The slides were washed three times in PBS and mounted in Cititluor (City University, London, UK).

The number of pyknotic nuclei per section was determined as an avera.ge of the number of pyknotic cells counted in five optic nerve sections per animal. Only clearly condensed or fragmented nuclei were counted as pyknotic. Most pyknotic nuclei measured 2-4 pm in diameter, which was considerably smaller than the thickness of the section (8 pm).

Immunofluorescence staining of cryostat sections and cells in culture was performed as described previously [ 11.

Delivery of exogenous CNTF into the optic nerve The cDNA for the entire coding region of rat CNTF (600 bp) was ligated 3’ of a cDNA fragment encoding the first 20 amino

acids of a mouse NGF, as described previously [45]. This con- struct cvaz cloned into the pRcCMV expression vector (InVit- roGen). Transfection of the human embryonic kidney cell line 293 was performed by the calcium phosphate method and SW

bly transfected clones were selected with G418 for neomycin resistance. Single clones were picked and grown up and the supematants of these clones were screened for their ability to support the survival of ES chick ciliary neurons. As previously described for mouse D3 embryonic stem cells, more than 1000 units (corresponding to at least 1 ng of CNTF) were detectable per 1 ml of supematant from confluent cultures of these cells, indicating that they were able to synthesize and secrete CNTF efficiently. The stably transfected 293 cells secreting CNTF were transplanted into the subarachnoid space of postnatal rats, as described previously 11,461.

Delivery of exogenous ICF-7 into the optic nerve A synthetic analogue of human IGF-1, [Gln3, Ala*, Ty@, ~eul6]IGF-1 (IGF252), which has 600-fold reduced affinity for serum IGF-1 binding proteins but is equipotent to hIGF-1 at binding to IGF-1 receptors [13], was expressed ,and secreted from COS-7 cells that had been transiently tranfected with the plasmid PHYKhIGF252. This plasmid was constructed by in- serting the 1.65 kb BarnHI-SmaI fragment of plasmid pUB252b (containing the ZGF252 gene inserted into exon II of the bovine growth hormone (6GH) gene; obtained from M Bayne, Merck Sharpe & Dohme, Inc., Rahway, New Jersey, USA) into the

4% Current Biology 1993, Vol 3 No 8

HindII-SmuI backbone fragment of the vector PHYKA5 (obtained from W’D Richardson and constructed as described in [l]). The PHYKA5 fragment was prepared by replacing the Hi&II site of PHYKA5 with BmzHI and then .cutting with BurnHI and SmaI. The resulting IGF252 expression plasmld con- tains the%GH-fGF252 fusion gene, which expresses the secre- tory sequence of bGH fused to IGF252, downstream of the ade- novirus major late promoter. The COS-7 cells were transfected and implanted into the subamchnoid space of postnatal rats, as previously described [ 11.

Acknmu1edgement.s~ We thank M Shipman for help with confocal microscopy. BAB is supported by a fellowship from the British Multiple Sclerosis Society.

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Receive& 27 May 1993; revised: 23 June 1993. Accepted: 23 June 1993.

Does oligodendrocyte survival depend on axons?

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