Journal of Neuroscience Research 26:409418 (1990)

Axons Modulate Myelin Protein Messenger RNA Levels During Central Nervous System Myelination In Vivo G. J. Kidd, P.E. Hauer, and B.D. Trapp Department of Neurology, Johns Hopkins University School of Medicine, Baltimore

Expression of myelin protein genes by myelinating era1 and central nervous systems (PNS and CNS, respecSchwann cells in vivo is dependent on axonal influ- tively). In part, different patterns of myelin gene ences. This report investigated the effect of axons on expression reflect differences in myelin composition and myelin protein mRNA levels in the central nervous hence those genes that are active during myelination. For system (CNS). In situ hybridization studies of rat spi- instance, proteolipid protein (PLP) accounts for approxnal cord sections localized mRNAs encoding proteo- imately 50% of CNS myelin protein (Norton and Camlipid protein (PLP) and myelin basic protein (MBP) mer, 1984) but is found only in minute quantities in 20 and 40 days after unilateral rhizotomy. Compared peripheral nerve and appears to be restricted to the with control tissue, hybridization intensity was re- Schwann cell cytoplasm (Puckett et al., 1987; Griffiths duced in transected tissue, but there was little change et al., 1989), whereas the major PNS myelin glycoproin the number of oligodendrocytes labeled. Cellular tein Po has not been detected in the CNS (Lees and RNA was extracted from transected and age-matched Brostoff, 1984). Another important phenotypic differcontrol optic nerves 5, 10, 20, and 40 days after sur- ence between Schwann cells and oligodendrocytes is that gery, and levels of the following mRNAs were deter- myelinating Schwann cells ensheath a single axon and mined by slot blot procedures: PLP, MBP, myelin- form one myelin internode along it, whereas individual associated glycoprotein (MAG), and 2’,3’ cyclic oligodendrocytes have the potential to myelinate multinucleotide 3’-phosphodiesterase (CNP). In transected ple axons and must match the production of myelin comnerves, PLP and MBP mRNA levels were approxi- ponents to the requirements of synthesizing multiple mately 85%, 45%, and 25% of control values at 5, 20 sheaths (Blakemore, 1981). In the PNS, the axon is the major regulator of myand 40 days posttransection, respectively. Axonal transection had a lesser effect on CNP and MAG elin gene expression. The experiments of Weinberg and mRNA levels, which declined to approximately 60% Spencer ( 1975, 1976) and Aguayo et al. ( 1976) provided of control levels at 40 days. Immunocytochemical evidence that all Schwann cells have the potential to studies indicated that the number of oligodendrocytes form myelin. However, for significant expression of mywas not decreased 40 days after optic nerve transec- elin genes either in vivo or in tissue culture, the Schwann tion. These data demonstrate that axons modulate cell first requires contact with an appropriate axon myelin protein mRNA levels in oligodendrocytes. In (Weinberg and Spencer, 1975, 1976; Aguayo et al., contrast to Schwann cells, however, oligodendrocytes 1976; Brockes et al., 1980; Mirsky et al. , 1980; Politis et continue to express significant levels of myelin protein al., 1982). Myelinating Schwann cells respond to degeneration of the ensheathed axon by rapidly down-regumRNA in vivo following loss of axonal contact. lating the synthesis of the myelin components such as Po Key words: optic nerve transection, in situ hybrid- and myelin basic protein (MBP; Politis et al., 1982; Poization, myelin protein mRNA levels, GFAP mRNA duslo, 1984). Recent studies investigating the mechalevels, immunocytochemistry nisms of myelin protein gene regulation have demonstrated a 40-fold decrease in Po and MBP mRNA levels INTRODUCTION Although the myelin sheaths of oligodendrocytes and Schwann cells perform similar functions and share many ultrastructural features, aspects of myelin formation and maintenance differ considerably in the periph0 1990 Wiley-Liss, Inc.

Received January 26, 1990; accepted February 5 , 1990. Address reprint requests to G.J. Kidd, Department of Neurology, Johns Hopkins University School of Medicine, Meyer 6-181, 600 N . Wolfe St., Baltimore, MD 21205.

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5 days after transection of actively myelinating rat sciatic nerve (Trapp et al.. 1988b). Posttranslational mechanisms of myelin protein regulation in Schwann cells are also believed to be important (Poduslo, 1984, 1985; Poduslo et al., 1985). In contrast, oligodendrocytes cultured in the absence of axons express significant levels of MBP mRNA (Zeller et al., 1985); synthesize myelin components such as galactocerebroside, MBP, PLP, and myelin-associated glycoprotein (MAG); and elaborate myelin-like membranes (Mirsky et al., 1980: Abney et al., 1981; Ranscht et al., 1982; Bradel and Prince. 1983; Zeller et al., 1985; Dubois-Dalcq ct al., 1986). This demonstrates that axonal contact is not required for myelin gene cxprcssion in oligodendrocytes. Nevertheless, the strong correlation between axonal diameter and myelin \heath thicknessiinternodal length in adult animals (McDonald and Ohlrich, I97 1 ; Hildebrand and Hahn, 1978; Murray and Blakemore, 1980; Blakemore, 198 1) suggests that axons play a role in modulating the synthesis of myelin components by oligodendrocytes. The present study investigates axonal modulation of myelin protein mRNA levels in the CNS following axonal transcction. The numbers of oligodendrocytes expressing PLP and MBP mKNA and the distribution of mRNA in these cells were determined by in situ hybridization in the rat spinal cord after unilateral dorsal and ventral rhizotomy. Myclin protein mRNA levels were quantitated in rat optic nerve after surgical transection, because this CNS tissue could be isolated completely from nontransected tissue. Levels of mKNAs encoding the CNS myelin proteins PLP, MBP, MAG and 2',3' cyclic nucleotide 3'-phosphodiesterase (CNP) and, for comparison, astrocyte glial fibrillary acidic protein (GFAP) were determined. Kcductions in myelin protein mRNA levels in oligodendrocytes after axonal transcction provide direct evidence that axons modulate myelin gene expression by 01 igodendrocytes in vivo. MATERIALS AND METHODS Animal Surgery and Tissue Preparation Male Sprague-Dawley rats (21 days old) were anesthetized, the right sciatic nerve was cut, and the L4 and L5 dorsal and ventral roots were removed. These rats were allowed to survive for 20 and 40 days after surgery and then perfused with 4% paraformaldehyde in 0.08 M phosphate buffer. The spinal cord was removed and the L4 and L5 regions were processed to paraffin by standard procedures. Serial sections (6 p m thick) were cut and alternate sections processed for either in situ hybridimtion or immunocytochcmistry, as described below. The right optic nerve of 21 -day-old male SpragueDawley rats was exposed in the orbit and transected 1-2

mm from the eye. The eye was rotatcd to revcal the proximal stump to verify complctc ncrve transcction in each case. Groups of 40 rats were subsequently sacrificed at 5 , 10, 20, and 40 days posttransection and left (intact) and right (transected) optic nerves were obtained, rapidly frozen, and stored at -70°C until required. Optic nerves from untreated rats were obtained at 7, 14, 21, 26, 3 1 , 41, and 61 days of age to provide control RNA satnples. In addition, rats at each time point after optic nerve transection were perfused with 4% paraformaldehyde. Segments from the middle third of each optic nerve were cryoprotected with 2.3 M sucrose and 30% polyvinyl pyrrolidole, mounted on specimen stubs, and frozen in liquid nitrogen. Cryosections (1 p m thick) wcrc cut in a Reichert Ultracut EIFC-4 cryo-ultramicrotome, transferred to glass slides coated with 3-aminopropyltriethoxysilane (Sigma), and stained with CNP antiserum as described hclow.

In Situ Hybridization Spinal cord sections were deparaffinized, rehydrated, and treated in 0.2 M HCI and protcinase K as previously described (Trapp et a]., 1987). Sections were hybridized for 16 hr at room temperature with 3sS nicktranslated cDNA probes complementary to MBP or PLP, washed at room temperature in 1 X SSC (0.03 M NaCl, 0.03 M sodium citrate, pH 7.4) followed by 50% formamide (Fluka), and dehydrated in ethanol containing 0.3 M ammonium acetate. Slides were dipped in NTB-3 nuclear track emulsion (Kodak) and processed 1 week later by standard autoradiography protocols (Trapp et al., 1987). After lightly counterstaining with haematoxylin, the sections were examined and photographed in a Zeiss Axiomat microscope using bright-field and dark-field optics. The number of grains was counted over individual cells (PLP) or within standard-sized ficlds (MBP) in control and transected tissues, and means and standard crror values were calculated. Immunocytochemistry Paraffin scctions of spinal cord, adjacent to those used for in situ hybridization, were deparaffinized and rehydrated. After blocking with 3% normal serum, these tissues were incubated with either polyclonal MBP antiserum raised in goat (1:250) or rabbit PLP antiserum (1 :250) and stained by the peroxidase antiperoxidase method as described previously (Trapp el al., 1987). Cryosections ( 1 pm thick) of transected and control optic nerves were immunostained with affinity-purified CNP antiserum (Trapp et al., 1988a) by the avidin-bi0th-peroxidase complex method (Vector ARC Elite

Myelin Protein mRNA Levels

Kit). The numbers of stained and unstained cells in each section were counted using Nomarski optics at x 63.

RNA Isolation and Analysis Optic nerves were homogenized in cold 4 M guanidine isothiocyanate containing 0.7% P-mercaptoethanol and total cellular RNA was extracted by cesium chloride ultracentrifugation as described by Maniatis et al. (1982). 'Typical yields from 40 optic nerves were 15-25 p g RNA. After phenol, chloroform, and ethanol washes (Maniatis et al., 1982). 1 p g RNA samples were denatured by boiling in 28.5% formamide (Flukaj, 1.25% formaldehyde, 5.7 mM Tris and then slot-blotted onto nitrocellulose rilters (Schleichcr and Schuell) in 10 X SSC containing 10 p g yeast tRNA per sample. In addition to optic nerve RNA, samples containing 0.25, 0.5, I , 1.5, 2, 2.5, 3 , 4, 5, and 7 p g spinal cord RNA were included on each blot to provide a standard series of samples having known relative RNA concentrations. Rat liver and kidney RNA samples (5 k g j were also used on each blot to control for nonspecific RNA-DNA interactions. cDNA probes were labeled by nick-translation (Bohringer Mannheim Nick-Translation Kit) using "P-ATP. The following well characterized cDNAs were used: PLP (Milner et al., 19&5),MBP (Roach et al., 1983), CNP (Bernier et al., 1987), MAG (Salzer et al., 1987), and GFAP (Lewis et al., 1984). Prior to hybridimtion, the nitrocellulose blots were baked at 80°C for 2 hr to immobilize the RNA, then preincubated in a hybridization mixture containing 50% formamide, 5 X SSC, 5 X Denhardt's Solution (Maniatis et al., 1982), 0.1% sodium pyrophosphate, and 0.1% SDS. Blots were hybridized overnight at 42°C and then washed in 2 X SSC, 0.15% sodium pyrophosphate, and 1 % SDS, then in 0.1 x SSC and 0.1% SDS. Dried nitrocellulose blots were used to expose Kodak X-omat film, and the resulting autoradiograms were quantitated by densitonietry using an LKB 2202 densitonieter and 2220 integration unit. A standard curve relating densitometer hybridization signal values and relative mRNA concentrations was drawn for each blot from the series of spinal cord dilutions. Rclativc mRNA levels for optic nerve samples were then obtained from integrated densitometer values using this curve. Optic nerve RNA samples were also analyzed by electrophoresis on 1% agarose gels containing 6.6% formaldehyde (Maniatis et al., 1982). Samples of 2 p g RNA/lane were denatured by boiling in 50% formamide, 6.6% formaldehyde in MEA buffer (Maniatis et al., 1982) and applied to the gel. Ribosomal RNA markers wcre run in adjacent lanes. RNA was immobilized on Genescreen (New England Nuclear) by Northern transfer (Maniatis et al., 1982), and hybridized with 32P-labeled

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TABLE I. Relative In Situ Hybridization Labeling Intensity of Spinal Cord Dorsal Columns After Spinal Root Transection* Days after transectian 20 40

Percent o f control values MRP

PLP

45.8 44.2

34 38

*I,aheling indices are expressed as a percent of values from the control dorsal column counted over laheled cells (PLP) or per unit area (MBP).

cDNA probes as dcscribed above, but with 1% SDS in the hybridization mixture.

RESULTS MBP and PLP In Situ Hybridization in Spinal Cord After unilatcral transection of the sciatic nerve and removal of the L4 and L5 ventral and dorsal roots, MBP and PLP immunostaining indicated myelin degeneration in the right dorsal column of the spinal cord at levels L4 and L5 (Figs. 1 A, 2A). Fibers in the adjacent contralatera1 column showed no evidence of myelin degeneration. The contralateral dorsal column provided a useful control tissue with which to compare transected myelin protein mRNA levels after in situ hybridization, particularly since both transected and control tissues were sectioned and hybridized under identical conditions. Similar patterns of silver grain distribution were observed in transected and control columns after in situ hybridization (Figs. I , 2), although signal intensity was considerably reduced in transected tissue. PLP labeling appeared characteristically as clusters of grains that were mainly restricted to the perinuclear regions when observed with bright-field microscopy (Figs. 2D,E) as previously reported (Trapp et al., 1987;Jordan et al., 1989). Because of this labeling pattern, it was possible to quantitate grain numbers over labeled cells following nerve transection in reprcsentative fields that appearcd free from intact myelinated fibers in adjacent immonostained sections (Table I). Cells were considered labeled if there were morc than seven grains over them; this criterion was sufficient to exclude endothelial and meningeal cells. The labeling index for cells in the transected dorsal column at 20 and 40 days was about one-third that of controls (see 'Table I). The number of grains over individual labeled cells in the transected tissue was fairly uniform (33.8 3.2 [SEMI in transected, 89.3 2 10.9 [SEMI in control at 40 days), so there was no evidence of subpopulations of oligodendrocytes with high and low levels of expression. At 40 days posttransection, there was little difference in the actual number of labeled oli-

*

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viously described (Trapp et al., 1987). In the transected side. silver grains appeared less evenly scattercd and often appeared as small clusters around cell nuclei (Figs. 1, 2). The average number of grains within representative fields of the transected dorsal column after MBP hybridization was less than one-half (see Table I) that of equivalent regions in the contralateral control side.

Myelin Protein mRNA Levels The developmental patterns of myelin protein mRNA levels in intact optic nerves from untreated control groups are presented in Figure 3. Relative mRNA levels were determined by densitometry of slot blot autoradiograms and using a reference dilution curve (see Matcrials and Methods). MBP, PLP, CNP, and MAG mRNA were highest at 14 days. After declining from 14 day values, MBP and PLP mRNA levels remained relative constant between 21 and 41 days and then decreased to 65% of 21 day levels at 61 days of age. CNP and MAG mRNA levels fell considerably between 14 and 21 days and declined further at 26 days but then remained relatively constant. These data indicate that during the period of the transection study, from 21 to 61 days of age. the effects of axonal transection are not superimposed on major developmental fluctuations in myelin protein mRNA levels. In contrast, GFAP mRNA levels were initially highest at 7 days, then declined at 14 and 21 days (data not shown). Levels remained relatively constant at 21, 26. and 31 days, then increased to reach 180% of 21 day levels at 61 days. Relative levels of myelin protein mRNAs were determined 5, 10, 20, and 40 days after right optic nerve transection (corresponding to 26, 3 1, 41, and 6 1 days postnatally) and are presented in Figure 4. The declines in MBP and PLP mRNA levels were similar relative to those of age-matched controls. Levels were reduced by 50% at 10 days, 60% at 20 days, and 70-8076 at 40 days. MAG mRNA levels were 65% of control values at Fig. I . Serial sections (6 Frn parallin) from longitudinally oriented rat spinal cord level L4 dorsal columns 20 days after 10 days and 60% at 40 days. In contrast to the 40% spinal root transection. The first section (A) was immuno- reduction in control nerves (see Fig. 3), CNP was restained with PLP antisera, and the subsequent sections, pho- duced by only 10% at 5 days after transection. Thus there tographed in dark-field, were hybridized with cDNA probes for was 33% increase in CNP levels compared with agePLP (B) and MBP (C). Myelin degeneration is extensive in the matched controls (Fig. 4D). At 10 and 40 days posttransected dorsal column (lower region), but the contralateral transection, CNP levels were 75% and 60% of control nontransccted column appears free from degenerating fibers. values, respectively. In contrast to myelin protein PLP and MBP hybridization signals are considerably reduced mKNA levels, GFAP mRNA levels (Fig. 5) increased in the corresponding transected areas of the adjacent sections. 2.4-fold 10 days after transection and were approxiBar = 25 pm. mately 3.5 times control levels at 20 and 40 days. Analysis of mRNA levels in the nontransected (left) optic nerves of treated rats provided evidence of a godendrocytes counted in areas of the same size in con- contralateral response following unilateral optic nerve transection (Fig. 6). PLP and MBP mRNA levels detrol and transected columns. MBP mRNA hgbridimtion signal was located dif- clined slightly in the contralateral nerve at 5 days postfusely throughout the neuropil of intact tissues, as pre- transection compared with levels in untreated age-

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Fig. 2. Transverse serial sections from dorsal columns 40 days after spinal root transection. The first section (A) was inimunostained for MBP. and thc two subscquent sections, photographed in dark-field, were hybridized for PLP (B) and MBP (C). In the transected dorsal column (left side in A-C), PLP and MBP hybridization signals are considerably reduced compared with the control region. At higher magnification, silvergrains representing PLP mRNA are clustered around nuclei

(arrowheads) in both control (D) and transected (E) dorsal columns. Fewer grains are present over labeled cells after transection compared with controls. Although more cells are present in the transected column as a response to axonal and myelin degeneration (macrophages/niicroglia, astrocytes), a similar number 01' labelcd cells is present (per unit area) in control and transected tissue. Bar in A-C = 100 p m , in D and E 25 pm.

matched controls (26 days postnatally). However at 10 days, mRNA levels were elevated by about 90% over control values (31 days) and were 5 0 4 5 % higher than untreated values at 40 days. Similar responses were also observed for CNP and MAG, although the magnitude of the 10 day peak was not as great. A steady increase in

contralateral GFAP mRNA levels was also observed after transcction. Northern blot analysis of RNA samples after transection indicated no differences in the apparent mobilities of the two major bands detected using probes complementary to PLP or for the major MBP band after

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200

and proportion of oligodendrocytes was similar in control and transccted optic nerves at all time points.

I A PLP

DISCUSSION

MBP CNP

MAG

09'

i

7

14

I

I

21 26 31 AGE (days postnatal) "

41

61

I

Fig. 3. Relative myelin protein mRNA levels during postnatal development in control optic nerves from untreated rats, determined from integrated densitometry scans of slot blot autoradiograms after hybridization with PLP, MBP. CNP, and MAG. Levels are expressed as a percentage of 21 day values. Each point represents the mean and SEM of three blots.

TABLE 11. Number of Oligudendrucytes in 1 prn Cryosections of Transected and Control Rat Optic Nerve After lrnmunostaining With CNP Antiserum* Days after transection 5 10

20 40

Control I %)

53 ( 1 70) 52 (170) 52 (127) 49(115)

Transxted

(Q)

46 (132) 46 (155) 49 (167) 51 (117)

"Data expressed as a percentage o f total cells in each section, with actual numher counted in parentheses.

transection. On slot blot autoradiograms, hybridization signals for 5 pg liver and kidney samples were rarely detectable at optimal exposure times for quantitation of 1 pg optic nerve samples.

Oligodendrocyte Numbers After Transection In 1 pin cryosections of both control and transected optic nerves, oligodendrocytes were distinguishable from astrocytes and macrophagesimicroglia by the presence of CNP staining deposit subadjacent to the perikaryal plasma membrane (Fig. 7). Using differential interference contrast (Normarski) optics, the nuclei of stained and unstained cells in single sections were identified at 5, 10, 20, and 40 days (Table 11). The number

The major aim of this study was to cstablish the extent of axonal modulation of myelin protein mRNA levcls in actively myelinating oligodendrocytcs. Our quantitative slot blot analysis demonstrates that PLP and MBP mKNA levels are reduced by similar degrees at 10 (50%),20 (65%), and 40 (75%) days after nerve transection. Levels of CNP and MAG mRNA were reduced to a lesser degree (40% at 40 days). Our in situ hybridimtion studies confirmed the reduction in PLP and MBP niRNAs and provide additional information about their cellular distribution. The distribution of PLP mRNA (and CNP immunostaining) demonstrates that the number of oligodendrocytes expressing myclin genes did not differ significantly from control nerves. We conclude therefore that axons modulate but do not totally regulate CNS myelin protein gene expression in vivo. The greater decline of PLP and MBP mRNA levels after optic nerve transection compared with values for CNP and MAG presumably reflects decreased cellular requirements for these two components of compact myelin, since axonal myelination is no longer possible, and preexisting myelin sheaths have mostly degenerated by 40 days (Hasegawa et al., 1988; Kidd, unpublished observations). By comparison, CNP is enriched beneath all oligodendrocyte plasma membranes, except compact myelin (Braun et al., 1988; Trapp et al., 1988a; Brunner et al., 1989): hence CNP mRNA would continue to be required despite the rcduced myelinating status of the oligodendrocyte after transection. The relatively small reduction in levels of MAG mRNA in transected tissues compared with changes for PLP and MBP mRNAs may reflect different pathways of regulation for this molecule from thosc of other myelin proteins. Recent studies have suggested that MAG is necessary for axonal recognition and contact (Poltorak et al., 1987; Trapp et al., 1989) and thus may be regulated by mechanisms independent of those governing the expression of genes required for subseyucnt myelin formation. Whereas major reductions were observed in myelin protein mRNA levels at 40 days after optic nerve transection, values for PLP and MBP were still approximately one-quarter those in age-matched controls; CNP and MAG levels remained much higher. These data are consistent with in vitro observations that myelin protein mRNA (Zeller et al., 1985) and myelin proteins (Mirsky et al., 1980; Abney et al., 1981; Bradel and Prince, 1983; Dubois-Dalcq et al., 1986) are synthesized by oligodendrocytes lacking axonal contact. This is in marked contrast with Schwann cells, in which a rapid reduction

Myelin Protein mRNA Levels C

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60

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20

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40

40

0)

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C

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83

80 -

=loo

T

5 10

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2

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60 -

2

2

140

0 120 -

?

-

Q

20

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m

a

100

2 80

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C

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60

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Fig. 4. Slot blots of myelin protein mRNA levels in transected oplic nerves (T) 5 , 10, 20, and 40 days after surgery and in control optic nerves (C) from untreated age-matched animals: after hybridization with PLP, MBP, MAG. and CNP probes.

Relative mRNA levels in transected nerves are plotted as a percentage of the appropriate age-matched control. Each point represents the mean and SEM of three blots.

in Po and MBP niRNA levels to 5% of those in controls were observed 5 days after sciatic nerve transection (Trapp et al., 1988b). Thus, although axons are required for normal expression of myelin genes in actively myelinating oligodendrocytes, significant levels of myelin protein rnRYAs are still detected in vivo in oligodendrocytes deprived of axons. Several possible mechanisms may rcgulate myelin protein mRNA levels in oligodendrocytes after nerve transection. Evidence has been presented by Sorg et al. (1987) suggesting that, in the absence of normal PLP gene expression in ,jimpy mice, MBP mRNA production is suppressed primarily through reduced rates of transcription. On the other hand, disparity bctween nuclear and cytoplasmic MBP and PLP mRNA levels during the development of normal mice (Sorg et al., 1987) and in cultured oligodendrocytes (Kumar et a]., 1989) suggests that mRNA turnover rates also play an important role in gene regulation. The yield of RNA in the present study

was not sufficient to determine whether reduced mRNA levels reflected decreased transcription rates or increased InRNA turnover. The translation efficiency of myelin protein mRNAs in oligodendrocytes may be inodified after axonal degeneration (Campagnoni et al., 1987), and the fate of translation products also remains to be determined. In vitro studies have indicated that oligodendrocytes are competent to synthesize PLP and MBP and incorporate them into myelin-like membranes in the absence of axons (Mirsky et al., 1980; Abney et al., I98 1 ; Bradel and Prince, 1983; Dubois-Dalcq et al., 1986). In vivo, oligodcndrocytc cell bodies in transectcd nerves are often surrounded by a small number of myelin lamellae (Fulcrand and Privat, 1977; Kidd, unpublished observations), indicating that oligodendrocytes transcribe and translate myelin gencs and incorporate proteins into myelin-like membranes after nerve transection. In contrast, little myelin gene transcription occurs in Schwann cells after nerve transection, myelin membrane assembly

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Fig. 5 . GFAP mRNA levels in transected optic nerves (T) 5, 10: 20, and 40 days after surgery and in control optic nerves (0 from untreated age-matched animals. Relative mRNA Ievels in transccted nerves are plotted as a percentage of agematched controls. Each point represents the mean and SEM of three blots.

Fig. 7. CNP immunostaining in 1 p m cryoscctions of control (A) and 20 day transected (B) rat optic nerves. In both control and transected tissues, oligodendrocytes are characterized by a

I

r

5

10

20

40

Days Atter Transection

Fig. 6 Contralatcral increase in relative myelin protein mRNA levelc in the intact (left) optic nerves of rats in which the right optic nerve was transected. Data plotted as a percent of values from age-matched untrcated rats, after hybridization with PLP, MBP, CNP, MAG, and GFAP cDNA probes

ring of staining deposit subadjacent to the perikaryal plasma membrane (arrowheads), whereas astrocytes (A) remain unstained. Bar = 20 prn.

Myelin Protein mRNA Levels

docs not occur, and translated proteins may be rapidly degraded (Poduslo, 1984, 1985; Poduslo et a]., 1985). Myelin protein mRNA lcvcls in the contralateral nontransected left optic nerve were increased at 10 days and subsequent time points compared with untreated agematched controls. GFAP mRNA levels also increased in the contralateral side after surgery, rising more steadily to reach levels about twice those of untreated controls I D days after transection. A possible explanation for these data is that elevated myelin protein mRNA levels in the left optic nerve may rcf lcct increased axonal rcquirements for myelination following synaptic reorganization. Approximately 10% of axons from each nerve do not cross over at the rat optic chiasm (cowcy and F’ranzini. 1979) but rather form synapses in the same target fields as those from the contralateral side. Degeneration of axons from the right optic nerve, therefore. may stimulate expansion of the terminal synaptic fields of remaining axons in the left optic nerve. Recent studies have indicated that such collatcral branching of axons results in increased axonal diameter and possibly stimulates an increase in myelin spiral length (Voyvodic, 1989). After axonal degeneration, inyelinating Schwann cells dediffercntiate to a premyelinating phenotype. which includes the suppression of myelin synthesis (Politis et al., 1982; Poduslo, 1984), the reexpression of down-regulated cell adhesion molecules (Jessen et al., 1987), and the production of nerve growth factor receptor (Taniuchi et al.. 1986). In contrast, oligodendrocytes maintain their myelin-forming phenotype and continue to synthesize myelin components and incorporate them into sheets of myelin-like membranes (Mirsky ct al.. 1980; Abney et al., 1981; Bradel and Prince, 1983; Zeller ct al., 1985; Dubois-Dalcq et al., 1986). In this study we have demonstrated that oligodendrocytes in vivo express significant, albeit reduced, levels of myelin protein mKNAs after axonal transection. These studies indicate that expression of myelin genes by Schwann cells and oligodendrocytes in vivo is regulated in part by different mechanisms.

ACKNOWLEDGMENTS We are grateful to Pamela Talalay for her help in preparing the manuscript. Thiq work wa\ supportcd by grant NS 22849 from the NIH and grant JF 2030 A-1 from the National Multiple Sclerosis Society. B.D.T. is a Harry Weaver Keurorcience Scholar of the National Multiple Sclerosis Society.

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