Journal of Neurocytology 13, 961-974 (1984)

Effects of neonatal transection on glial cell development in the rat optic nerve: evidence that the oligodendrocyte-type 2 astrocyte cell lineage depends on axons for its survival

S A M D A V I D * , R O B E R T H . M I L L E R t , R A M I L A P A T E L { a n d M A R T I N C. R A F F

Medical Research Council Neuroimmunology Project, Department of Zoology, University College London, London WCIE 6BT, UK

Received 14 June 1984; revised 3 August 1984; accepted 17 August 1984

Summary

We have previously provided evidence that the rat optic nerve contains three types of'macroglial cells that develop as two distinct lineages: one lineage comprises type I astrocytes, which develop before birth, while the other comprises oligodendrocytes and type 2 astrocytes, which develop after birth from a common, bipotential glial progenitor cell. In the present study we have examined the influence of axons on the development of these two glial cell lineages by cutting the optic nerve at birth so that the retinal ganglion cell axons in the nerve degenerate. Using antibodies to distinguish the different types of glial cells in suspensions and semithin frozen sections of cut and uncut optic nerves, we show that neonatal transection results in a striking decrease in the total number of oligodendrocytes, type 2 astrocytes and their progenitor cells but has much less effect on the number of type 1 astrocytes. Since the [3H]thymidine labelling indices of oligodendrocytes and their progenitor cells were not significantly decreased in cut nerves, our results suggest that the progenitor cells and/or their progeny die in large numbers following neonatal nerve transection. We conclude that axons are required for the survival of cells of the oligodendxocyte-type 2 astrocyte lineage, at least during postnatal development.

Introduction

The optic ne rve is an attractive par t of the ver tebrate C.N.S. for s tudy ing glial cell

deve lopment . For example , the d e v e l o p m e n t of astrocytes and ol igodendrocytes can be

*Present address: Division of Neurology, The Montreal General Hospital, Montreal, Canada H3G 1A4. tPresent address: Department of Anatomy, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA. :~Present address: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

0300-4864/84 $03.00 + .12 �9 1984 Chapman and Hall Ltd.

962 DAVID, MILLER, PATEL and RAFF

studied in the virtual absence of neuronal influence: either in vitro, since optic nerve cultures contain no neurons (Raft et al., 1983a), or in vivo, if the optic nerve is cut at birth so that the retinal ganglion cell axons degenerate. Several years ago, Fulcrand & Privat (1977) reported that very few oligodendrocytes developed in neonatally transected rat optic nerves, suggesting that oligodendrocyte development depends on axonal signals. Since they found that the proportion of astrocytes in cut nerves was increased, as was the proportion of glioblasts and oligodendrocytes that incorporated [3H]thymidine (Privat et al., 1981), they speculated that, in the absence of axons to induce them to become oligodendrocytes, glioblasts develop instead into astrocytes.

We have repeated these neonatal transection experiments in the light of two recent observations concerning glial cells in the optic nerve. The first is that two distinct subclasses of astrocytes can be distinguished in the rat optic nerve on the basis of their different developmental histories, locations in the nerve, antigenic phenotypes, and in vitro morphologies and growth properties (Raft et al., 1983a; Miller & Raft, 1984). Most type 1 astrocytes develop before birth (Miller et al., unpublished data), are found at the periphery of the adult nerve where they form the glial limiting membrane, and do not bind the monoclonal antibody A2B5 (Miller & Raft, 1984), which recognizes specific gangliosides (Eisenbarth et al., 1979). Most type 2 astrocytes develop after one week of age, are found in the interior of the adult nerve, and are labelled by A2B5 antibody (Miller & Raft, 1984). In the enucleation studies of Fulcrand & Privat (1977; Privat et aI., 1981), and indeed in all previous studies on glial cell development in the optic nerve (Vaughn & Peters, 1967; Vaughn, 1969; Kuwabara, 1974; Skoff et aI., 1976a, b; Valat et al., 1983), the astrocytes were considered as a single population.

The second observation that prompted us to re-examine the effects of neonatal optic nerve transection is one that suggests that glial cells in the rat optic nerve develop as two distinct lineages - one lineage giving rise to type I astrocytes beginning before birth and the other giving rise to oligodendrocytes and type 2 astrocytes after birth (Raft et al., 1983b, 1984). The critical finding here was the identification of a bipotential glial progenitor cell in the developing rat optic nerve that differentiates into an oligodendrocyte if cultured in serum-free medium and into a type 2 (but not type 1) astrocyte if cultured in 10% foetal calf serum (FCS) (Raft et al., 1983b). Type I astrocytes, on the other hand, develop from a different precursor cell, which can b e distinguished serologically from the oligodendrocyte-type 2 astrocyte (0-2A) progenitor celi as early as embryonic day 17 (E17) in the rat optic nerve (Raft et al., 1984).

Although it is clear that 0-2A progenitor cells can differentiate into oligodendrocytes or type 2 astrocytes in neuron-free cultures (Abney et al., 1983; Raft et al., 1983b), the findings of Fulcrand & Privat (1977) suggest that axons might influence the differentiation of these progenitor cells in vivo. They might do so in at least two ways. First, as suggested by Privat et al. (1981), they might normally induce oligodendrocyte differentiation and, in their absence following transection, most 0-2A progenitor cells might develop into type 2 astrocytes. Second, degenerating axons, resulting either from retinal ganglion cell death, which is known to occur in normal development (Lam et aI.,

Glial d e v e l o p m e n t in t ransected optic nerve 963

1982; Potts et aI., 1982), or f rom neonata l nerve transection, might posit ively induce 0-2A

progeni tor cells to deve lop into type 2 astrocytes. If axons influence glial cell

d e v e l o p m e n t in ei ther of these two ways , one would expect neonatal nerve t ransect ion

to result in a large increase in the ratio of type 2 astrocytes to o l igodendrocytes in the cut nerve.

To our surprise, this is not the result we obtained. Instead, neonata l optic nerve

t ransect ion resul ted in a str iking decrease in the number s of ol igodendrocytes , type 2

astrocytes and 0--2A progenitor cells but had much less effect on the number of type 1 astrocytes. Since the p ropor t ions of these glial cells and of the 0-2A progeni tor cells that

incorpora ted [3H]thymidine were not significantly decreased in cut nerves , the 0-2A

progeni tor cells and/or their p r o g e n y m u s t have died in large n u m b e r s in neonatal ly

t ransected nerves . This sugges ts that axons are required for the survival of cells of the

0-2A lineage, at least dur ing pos tna ta l deve lopment .

Materials and methods

Optic nerve transection One-day-old Sprague-Dawley rats were anaesthetized with ether and an incision was made in the region of the right palpebral fissure to expose the eye. The eye was gently retracted and the optic nerve was cut with microscissors just behind the globe. The incision was closed with 10-0 sutures. The left optic nerve was left intact to serve as a control. Approximately 150 operated rats were used in these studies, either 15 days or 60 days after the transection.

Cell suspensions and cultures Optic nerves from I day, 7 day or 15 day normal Sprague-Dawley rats, or from 15 day operated rats were dissected from just behind the eye to the chiasm. The nerves from approximately 25 operated rats were pooled for each experiment. They were cut into small pieces using iridectomy scissors and incubated for 20 min at 37 ~ C in 1 ml Minimal Eagle's Medium (MEM) with 0.02 M

Hepes buffer (MEM-Hepes) and an equal volume of 0.25% (w/v) trypsin (Difco) in Tris-buffered saline, pH 7.7, with collagenase (Worthington) added to a final concentration of 0.02% (w/v). After a second 20 min incubation in the same solution, the cells were incubated for 20 min at 37 ~ C in 1 ml trypsin solution and 1 ml 0.02% (w/v) EDTA in Tris-buffered saline with collagenase. The cells were then, dissociated in DNAase (Sigma, 0.04 mg ml -~) and trypsin inhibitor (Sigma, 0.05 mg m1-1) in Ca2+-free and Mg2+-free MEM by pipetting 15 times in a 2 ml graduated pipette, followed by syringing three times through a 25 gauge hypodermic needle on a 2 ml syringe. The resulting cell suspension was passed through a fine nylon mesh to remove debris and washed in 10 ml Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FCS (DMEM-FCS). The cells were counted in a haemocytometer in the presence of trypan blue.

In some experiments, dissociated cells from 15 day optic nerves, which had been transected at 1 day, were cultured for 3 days on poly-L-lysine (PLL)-coated glass coverslips in DMEM-FCS, as previously described (Raft et al., 1983b).

Frozen sections Semithin frozen sections of normal or transected optic nerves were prepared as previously described (Miller & Raft, 1984). Briefly, the optic nerves were fixed by immersion (in one-day-old

964 DAVID, MILLER, PATEL a n d RAFF

rats) or perfusion (in older rats) in 3% paraformaldehyde and 0.5% glutaraldehyde, cut into small pieces and immersed in 1 M sucrose with 2% paraformaldehyde for 1 h. The nerves were then mounted on the stub of a freezing microtome and frozen by immersion in a slush of Freon 22, which was cooled in a bath of liquid nitrogen. Semithin sections (0.25-0.5 ~tm) were cut using a Sorval MT2B microtome with an FTS cryoattachment on a 25 mm glass knife at -60 ~ C to - 70 ~ C.

Immunofluorescence labelling All of the antibodies used in these studies have been previously described. Three mouse monoclonal antibodies were used as ascites fluid: A2B5 (Eisenbarth et al., 1979), diluted 1:50-1:100; anti-galactocerebroside (GC) (Ranscht et al., 1982), diluted 1:100; and anti-neurofilament (RT97) (Wood & Anderton, 1981), diluted 1:50. Rabbit anti-glial fibrillary acidic protein (GFAP) (Pruss, 1979) antiserum was diluted 1:1000. The binding of monoclonal antibodies was detected with a rhodamine-conjugated goat anti-mouse immunoglobulin (G anti-MIg-Rd, Cappel, diluted 1:100), while the binding of the anti-GFAP serum was detected with a fluorescein-conjugated goat anti-rabbit Ig (G anti-Rig-F1, Nordic, diluted 1:100), which had been absorbed with mouse Ig coupled to Sepharose 4B until it no longer crossreacted with mouse Ig. In some experiments, the binding of anti-GC antibody was detected witlq fluorescein-conjugated goat anti-mouse IgG3 (G anti-IgG3-F1, Nordic, diluted 1:80).

Cell suspensions were double labelled with A2B5 and anti-GFAP antibodies, or with anti-GC and anti-GFAP antibodies as previously described (Raft et al., 1983a). In brief, cells were labelled in suspension with A2B5 or anti-GC antibody followed by G anti-MIg-Rd, then allowed to adhere to PLL-coated coverslips; after fixation in acid-alcohol, the cells were labelled with anti-GFAP antiserum followed by G anti-Rig-F1. To determine the proportion of A2B5 +, GC-, GFAP- cells, labelling was carried out so that A2B5 binding was visualized with rhodamine, and both anti-GC and anti-GFAP antibody binding were visualized with fluorescein: cells were labelled with A2B5 antibody followed by G anti-MIg-Rd, then anti-GC antibody followed by G anti-IgG3-F1, and finally, after acid-alcohol fixation, anti-GFAP followed by G anti-Rig-F1. Semithin frozen sections were labelled with anfi-GFAP serum or double labelled with A2B5 and anti-GFAP antibodies as previously described (Miller & Raft, 1984). Cells and frozen sections were examined in a Zeiss Universal incidence fluorescence microscope equipped with phase contrast, fluorescein and rhodamine optics. No significant staining was observed when normal ascites fluid or normal rabbit serum was used in place of the monoclonal antibodies or anti-GFAP serum, respectively. Specimens were photographed using Tri-X or Ektachrome colour slide film rated at 400 ASA.

Autoradiography Six-day-old rats, which had their right optic nerves transected at 1 day, were injected twice with 5 ~tCi per gram of body weight of methyl-[BH]thymidine (75 Ci mmo1-1, Amersham International) approximately 24 h and 16 h prior to sacrifice at 7 days. The transected and control nerves were removed and dissociated, and the resulting cell suspensions were labelled with A2B5 and anti-GFAP serum, anti-GC antibody, or with A2B5, anti-GC and anti-GFAP antibodies, as described above. The coverslips were coated with Ilford K5 emulsion, stored at - 70 ~ C for 6 weeks in the dark, developed with Ilford Super Contrast FF and finally examined and scored as previously described (Raft et al., 1983a).

Electron microscopy Sixty-day-old rats, in which one optic nerve was neonatally transected, were perfused with 3% paraformaldehyde and 0.5% glutaraldehyde. The optic nerves were removed and immersed in 3% paraformaldehyde and 1% glutaraldehyde overnight, then in 1% osmium tetroxide for 2 h,

Glial development in transected optic nerve 965

followed by 0.5% aqueous uranyl acetate for 4 h. Following dehydration through graded alcohols and embedding in Epon 812, transverse thin sections were cut and stained with uranyl acetate and lead citrate and then examined on a Jeol 100 CX II electron microscope.

Results

Cell suspensions Cells were dissociated from cut and control optic nerves 14 days after neonatal (P1) transection. The total number of cells was counted and the proportions of the different glial cell types were determined by immunofluorescence: oligodendrocytes were identified as galactocerebroside + (GC) cells, type 1 astrocytes as A2B5-, GFAP + cells, type 2 astrocytes as A2B5 +, GFAP-- cells, and 0-2A progenitor cells as A2B5 +, GC-, GFAP- cells (Raft et al., 1978, 1983a, b). The total number of each cell type was calculated by multiplying their proportion by the total number of cells obtained per nerve.

As can be seen in Table 1, the number of cells obtained from cut nerves was almost ninefold less than that obtained from uncut nerves from the same animals. There was a comparable decrease in the number of A2B5 +, GC-, GFAP- cells (presumptive 0-2A progenitor cells) and an even greater decrease in the numbers of oligodendrocytes and type 2 astrocytes, while the decrease in type i astrocytes was much less (Table 1). As was the case with normal nerves (Raft et aI., 1983b), no cells were found that were labelled by both anti-GC and anti-GFAP antibodies in suspensions of transected nerves.

To confirm that at least some of the A2B5 +, GC , GFAP cells in neonatally transected nerves were 0-2A progenitor cells, dissociated cells from such nerves were cultured in DMEM-FCS on PLL-covered glass coverslips for 3 days and then double labelled with A2B5 and anti-GFAP antibodies (Raft et al., 1983b). In such cultures, 15-25% of the cells were A2B5 +, GFAP + type 2 astrocytes (not shown), even though less than 0.1% of the cells in the starting population had this phenotype (Table 1). Since type 2 astrocytes divide very little in these cultures (Raft et al., 1983a, b), substantial numbers of type 2 astrocyte progenitor cells must have been present in cut nerves at 15 days.

Only 50-55% of the cells in suspensions of control nerves and only 4045% of the cells in cut nerves could be identified as astrocytes, oligodendrocytes or putative 0-2A progenitor cells by antibody labelling (Table 1). While we did not attempt to identify the other cells, it seems likely that they were mainly a mixture of leptomeningeal, endothelial and microglial cells. We have found in another study (Miller et al., unpublished data) that many of the astrocytes in 1t- 7 day rat optic nerve are not released into suspension by our dissociation procedure. Thus the total numbers of cells and the proportions of the different types of glial cells in such cell suspensions cannot be assumed to reflect the situation accurately in the intact nerve. For this reason, we compared the numbers of glial cells in semithin frozen sections of cut and normal optic nerves.

Semithin frozen sections As can be seen in Table 2, the total number of cells per complete cross-section of nerve

966 DAVID, MILLER, PATEL and RAFF

was at least threefold less in cut compared to normal nerves at 15 days. Moreover , cut nerves usually failed to grow in length following transection and were, therefore, less than half the length of control nerves on average at 15 days (Table 2). Thus, t ransection resulted in almost an eightfold loss in cell number , confirming the results obtained in cell suspensions.

When frozen sections were double labelled at 15 and 60 days with A2B5 and

anti-GFAP antibodies, there were striking differences be tween cut and normal nerves. While the amoun t of GFAP was much greater in cut nerves, the amount of A2B5 was much greater in normal nerves (Fig. 1). The increase in GFAP in cut nerves was due both to an increase in the propor t ion of astrocytes (Table 2) and to an increase in the amoun t of GFAP in each astrocyte (Fig. 1). The decrease in A2B5 in cut nerves was mainly due to the absence of A2B5 + axons, which was confirmed by electron microscopy (Fig. 2) and by immunofluorescence staining with anti-neurofi lament ant ibody (not shown). In addition, however , there were very few A2B5 + glial cells in cut nerves compared to

normal nerves. This was most apparent at 60 days, where most of the GFAP-- cells in normal nerves were A2B5 +, while only rare A2B5 +, GFAP-- cells were seen in cut nerves (Fig. 1). Thus the decrease in numbers of type 2 astrocytes in cut nerves compared to normal nerves was very much greater than the decrease in type i astrocytes. Moreover , at 15 days more than 20% of the cells in cross-sections of normal nerves were A2B5--, GFAP- , while only about 5% had this pheno type in cut nerves (Table 2). Thus there was more than a 20-fold decrease in such putative 0-2A progenitor cells in cut nerves compared to normal nerves.

Al though we were unable to identify ol igodendrocytes directly in frozen sections, it is likely that they const i tuted most of the cells that were not labelled by A2B5 or anti-GFAP

antibodies in the normal nerves. The finding that more than 70% of the cells in the 15 day cut nerves were GFAP + astrocytes (Table 2) means that less than 30% could have been ol igodendrocytes in such nerves. In fact, many of the A2B5-, GFAP- cells in such nerves contained cell debris and were probably macrophages (Fulcrand & Privat, 1977).

Since the total number of cells was decreased almost eightfold in cut nerves (Table 2) and the propor t ion of recognizable ol igodendrocytes in normal 15 day rat optic nerve has been repor ted to be greater than 30% (Vaughn, 1969; Fulcrand & Privat, 1977), there must have been more than an eightfold decrease in ol igodendrocytes in cut nerves

Fig. 1. Semithin transverse frozen sections through a normal (A-C) and neonatally transected (D-F) optic nerve from a 60-day-old rat. The sections were labelled sequentially with A2B5 (B, E) and then anti-GFAP (C, F) antibodies as described in the text and were visualized under phase contrast (A, D), rhodamine (B, E) and fluorescein (C, F) optics. In the normal nerve (A-C), astrocyte nuclei are marked with an asterisk and several astrocyte processes are indicated with large arrows. Note that the fibrous astrocytes in the interior of the normal nerve are A2B5 + and GFAP +, while the protoplasmic astrocyte (two small arrows) at the periphery of the nerve is GFAP + but A2B5-. In the cut nerve, there are no A2B5 + cells (E) but the amount of GFAP staining (F) is much greater than in the normal nerve (C). Two blood vessels in the cut nerve are indicated with large arrows. Scale bar: 20 ~m.

Tab

le 1

. G

lial

cel

ls i

n s

usp

en

sio

ns

of

15 d

ay

op

tic

ne

rve

s af

ter

ne

on

ata

l tr

an

sec

tio

n.

Type

1

Type

2

Tota

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Olig

oden

droc

ytes

as

troc

ytes

as

troc

ytes

A

2B5

+,

GC

,

GF

AP

pe

r ne

rve

(GC

+)

(A2B

S-,

GF

AP

+)

(A2B

5 +

, G

FA

P +

) ce

lls

No

rma

l n

erv

es

42 3

00

+

10 8

00

11 5

50 •

15

10

1540

+

64

0 87

+

18

9325

+

18

00

Cu

t n

erv

es

4850

•

1250

41

0 +

95

72

4 +

14

5 <

5

930

+

380

The

nu

mb

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of s

peci

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cell

s w

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s m

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Tab

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. G

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in f

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s o

f 15

day

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Leng

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, G

C-,

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AP

- (m

m)

per

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(A2B

5-,

GF

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+)

(A2B

5 +

, G

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P +

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No

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es

6.8

+

0.3

33

8 +

11

62

•

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1.5

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(2

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(4

21)

(34)

(5

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3 +

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_+ 9

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Sec

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; co

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and

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. T

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um

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each

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l ty

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by t

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and

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give

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e.

Glial deve lopmen t in t ransected optic nerve 969

Fig. 2. Electron micrographs of transverse, conventional thin sections through a normal (A) and neonatally transected (B) optic nerve from a 60-day-old rat. While the normal nerve contains many myelinated axons (a) and relatively few astrocytes (ast), the transected nerve contains no myelinated axons and is composed largely of astrocytes. Scale bar: 5 ~m.

compared to normal nerves. In electron micrographs of cut nerves studied 60 days after neonatal transection, only occasional ol igodendrocytes were seen among the dense meshwork of type 1 astrocyte processes that made up the bulk of the nerve (Fig. 2B).

Autoradiography In order to s tudy the influence of optic nerve transection on glial cell division, animals in which one optic nerve was cut at 1 day were injected with [3H]thymidine at 6 days. The next day their optic nerves were dissociated and the cells were labelled with A2B5 and anti-GFAP antibodies, with anti-GC antibodies, or with A2B5, anti-GC and anti-GFAP antibodies (as described above) before being studied by autoradiography. The 6 day time point was chosen because the efficiency with which one can dissociate cells from optic nerve falls off rapidly after this age (Miller et aI., unpubl ished results). As can be

970 DAVID, MILLER, PATEL and RAFF

Table 3. Effect of neonatal transection on [3H]thymidine incorporation into glial cells in suspensions of 7 day optic nerve studied by autoradiography.

Proportion (%) of cells incorporating [3H]thymidine

Type 1 Oligodendrocytes astrocytes A2B5 +, GC-, GFAP- (GC +) (A2B5-, GFAP +) cells

Normal nerves 2 +.1 9 + 3 17 + 2 Cut nerves 3 + 1 8 + 4 15 _+_ 3

Six-day-old rats, which had one optic nerve cut at 1 day, received [3H]thymidine (5 ~Ci g 1) 24 h and 16 h prior to sacrifice at 7 days. Cell suspensions were labelled with A2B5, anti-GC and anti-GFAP antibodies and processed for autoradiography as described in text. Cells with /> 5 silver grains over their nucleus were counted as radiolabelled. Results are expressed as means _+ S.D. of three separate experiments.

seen in Table 3, the propor t ions of radiolabelled ol igodendrocytes, type i astrocytes and A2B5 +, GFAP- , G C - cells were not significantly different in cut and uncut nerves. There were no type 2 astrocytes at this age.

Discussion

We have found that neonatal optic nerve transection in rats leads to a large decrease in the total numbe r of glial cells in cut nerves compared to normal nerves at 15 days. However , the decrease does not affect all glial cells to the same extent. Using antibodies to distinguish the different types of glial cells in suspensions and semithin frozen sections of optic nerve (Raft et al., 1983a, b; Miller & Raft, 1984), we found that, while total cell numbers were decreased almost eightfold by neonatal transection, A2B5-, GFAP + type 1 astrocytes were decreased less than twofold. On the other hand, the numbers of A2B5 +, GFAP + type 2 astrocytes, GC-- ol igodendrocytes, and their common

A2B5 § GC - , GFAP- progeni tor cells were decreased more than eightfold. Thus, the gliosis that results f rom neonatal optic nerve transection is almost entirely a response of type 1 astrocytes, which perhaps could have been anticipated since they are the only astrocytes present at the time of transection. However , we have previously shown that reactive astrocytes dissociated from adult rat corpus callosum 5 or 8 days after a stereotaxic lesion also have the antigenic pheno type of type 1 astrocytes (Raft et al., 1983a) and m o r e recently have found that the same is true for the astrocytes in glial scars formed following adult optic nerve transection (Miller et al., unpubl i shed results). It seems that reactive gliosis in the C.N.S. is generally a function of type I rather than type 2 astrocytes. Our findings and those of Privat et al. (1981) that the [3H]thymidine labelling indices of type 1 astrocytes were not significantly different in normal and cut nerves, taken together with the observed decrease in the numbers of these cells in cut

Glial development in transected optic nerve 971

nerves, suggests that the gliosis in neonatally transected optic nerves does not involve enhanced astrocyte proliferation.

We have previously provided evidence for two distinct lineages of glial cells in the rat optic nerve - one comprising type 1 astrocytes, which develop before birth, and the other comprising oligodendrocytes and type 2 astrocytes, which develop from a common (0-2A) progenitor cell after birth (Raft et al., 1983a, b, 1984; Miller & Raft, 1984). Our present observations suggest that neonatal optic nerve transection dramatically depletes the second lineage while relatively sparing the first. Perhaps this is not surprising since, at the time of transection, most type 1 astrocytes have already developed while oligodendrocytes and type 2 astrocytes have not (Miller et al., unpublished results).

Why do so few oligodendrocytes and type 2 astrocytes develop in a neonatally transected optic nerve? In principle, there are at least four possible explanations, which are not mutually exclusive: (1) axons are required for the 0-2A progenitor cells to differentiate into oligodendrocytes and type 2 astrocytes; (2) axons normally provide mitotic signals for these glial cells and/or their progenitors; (3) degenerating axons positively kill these glial cells and/or their progenitors; (4) these glial cells and/or their progenitors are dependent on axons for their survival during development.

A number of findings argue against the first two explanations. If the first were correct, we would have expected an accumulation of 0-2A progenitor cells in the cut nerve, but instead, the number of these cells was greatly reduced. Moreover, this explanation does not account for the large overall loss of cells in neonatally transected nerves. Nor does it fit with the observation that 0-2A progenitor cells in perinatal optic nerve are able to differentiate into either oligodendrocytes or type 2 astrocytes in vitro in the absence of viable neurons (Abney et al., 1983; Raft et aI., 1983b). Our experiments and those of Privat et al. (1981) make the second explanation unlikely as the [3H]thymidine labelling indices of oligodendrocytes and 0-2A progenitors were not found to be significantly decreased in transected nerves compared to normal ones. The third explanation also seems highly unlikely since, to our knowledge, there is no precedent for a dying cell in a multicellular organism killing its normal neighbours. That leaves us with the fourth explanation, which we think is most likely to be correct - that is that the cells of the 0-2A lineage depend on axons for their survival during development, and when the axons are cut at birth, most of these cells die.

Not all of the cells of the 0-2A lineage died in neonatally transected optic nerves. For example, there were at least several hundred GC + oligodendrocytes in each cut nerve at 15 days (Table 1). Since GC + oligodendrocytes are not detectable at 1 day (Miller et aI., unpublished results), when the nerves were cut, it is clear that at least some 0-2A progenitor cells in 1 day optic nerve were able to differentiate in vivo into GC + oligodendrocytes in the virtual absence of viable axons. While we cannot exclude the possibility that the differentiation and survival of these cells depended on small numbers of residual axons (projecting from brain to retina), we were unable to detect such axons with anti-neurofilament antibodies or by electron microscopy. Moreover, we have previously found that 0-2A progenitors in E18'rat optic nerve can differentiate in

972 DAVID, MILLER, PATEL and RAFF

GC + oligodendrocytes in culture in the absence of detectable neurons (Abney et al., 1983). Thus, at the moment, there is no convincing experimental evidence that axons induce 0-2A progenitor cells to differentiate into oligodendrocytes.

We were surprised to find that, in transected optic nerves, even fewer type 2 astrocytes developed than oligodendrocytes. Even at 60 days, when the majority of GFAP + astrocytes in normal nerves were A2B5 § only rare A2B5 +, GFAP + cells were found in cut nerves, although more than 70% of the cells in cut nerves were GFAP § astrocytes. This finding is difficult to reconcile with the proposal of Privat et al. (1981) that in the absence of axonal signals to induce them to become oligodendrocytes, glial precursor cells develop into astrocytes. It also tends to exclude our unlikely suggestion (Raft et al., 1983b) that degenerating axons (for example, those resulting from retinal ganglion cell death during normal development: (Lain et al., 1982; Potts et al., 1982) may induce 0-2A progenitor cells in vivo to become type 2 astrocytes rather than oligodendrocytes.

What then determines whether an 0-2A progenitor cell normally develops into an oligodendrocyte or a type 2 astrocyte in vivo? While our experiments suggest that the 0-2A lineage largely depends on axons for its survival, they provide no evidence that axons play a part in the 0-2A progenitor cell's choice of differentiation pathway. The fact that oligodendrocytes develop before type 2 astrocytes (Miller et al., unpublished results) suggests the possibility that 0-2A progenitor cells differentiate into type 2 astrocytes only after a sufficient number of oligodendrocytes have been produced to ensure adequate myelination. But how such a scheme would be controlled remains to be determined.

We have previously found that 0-2A progenitor cells in neuron-free cultures of neonatal optic nerve prematurely decrease their rate of proliferation and differentiate into type 2 astrocytes or oligodendrocytes, depending on whether they are cultured in the presence or absence of FCS, respectively (Raft et al., 1983a, b, 1984). While we initially suspected that it was the lack of neurons in the cultures that was responsible for this abnormal behaviour of the progenitor cells, our present experiments make this interpretation unlikely: 0-2A progenitor cells in neonatally transected nerves seemed to proliferate normally and did not differentiate prematurely (see Table 1) in the virtual absence of viable axons. If signals from axons are not the answer, what is missing from optic nerve cultures that is present in the intact and transected nerve? One possibility is that type I astrocytes, which constitute a much larger proportion of the cells in the intact (or transected) developing optic nerve than in cultures of such nerves, provide the relevant sigilal(s). This hypothesis is supported by the recent finding that purified type 1 astrocytes can keep 0-2A progenitor cells proliferating in serum-free cultures and prevent them from prematurely differentiating Noble & Murray, 1984).

Acknowledgements

We thank Dr M. Nirenberg for the A2B5 clone, Drs B. Anderton and J. Wood for the anti-GC antibody, and Anne Mudge for helpful comme.nts on the manuscript. SD was

Glial development in transected optic nerve 973

supported by a Canadian MRC Centennial Fellowship and RHM by a fellowship from the National Fund for Research into Crippling Diseases.

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Note added in proof

After this paper was accepted for publication, a colleague suggested an alternative explanation for our results - that blood vessel damage at the time of transection may have resulted in ischaemic necrosis and thereby the preferential death of cells of the 0-2A lineage. We subsequently examined 1 ~zm Epon sections of neonatally transected optic nerves, 1, 3, 5 and 7 days after transection, and found no signs of necrosis.