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Axolemma-enriched fractions isolated from PNS and CNS are mitogenic for cultured Schwann cells
Developmental Brain Research, 3 (1982) 295-299
295
Elsevier Biomedical Press
Short Communications
Axolemma-enriched fractions isolated from PNS and CNS are mitogenic for cultured Schwann cells
GEORGE H. DeVRIES, JAMES L. SALZER and RICHARD P. BUNGE
Department of Biochemistry, Medical College of Virginia, Richmond, VA 23298 and (J.L.S. anti R.P.B.) Departments of Anatomy and Neurobioh~gy, Washington University School of Medicine, St. Louis, MO 63110 I U.S.A.)
(Accepted October 12th, 1981) Key words: axolemma fractions - - cultured Schwann cells -- mitogenesis -- myelinated axons
Axolemma-enriched fractions isolated from myelinated axons of rat and bovine CNS and PNS will stimulate a qmescent population of cultured rat Schwann cells to proliferate, The mitogen in the axolemma-enriched fraction is not lost in the extensive washing used to isolate the membrane fraction and is sensitive to heat and trypsin, suggesting that it is an integral membrane protein or glycoprotein. The axolemma-enriched preparations should provide an adequate source of Schwann cell mitogen for solubilization and purification of this membrane bound mitogen. Schwann cell proliferation appears to be regulated by axonal contact. This is suggested by both the in vitro observation that Schwann cell proliferation is dependent on a mitogenic signal provided by growing neurons7, t~, and the in vivo observation that Schwann cell numbers in nerve trunks are proportional to the number o f nerve fibers present t. Tissue culture preparations utilizing separated, pure populations o f sensory neurons and Schwann cells tt demonstrate that a mitogenic signal can be provided to Schwann cells by intact bare sensory axons11, t~, that cell-cell contact is required 12, and that a microsomal fraction derived from cultured neurites can substitute for the intact axon 13. Membrane fractions derived from other cell types in tissue culture did not stimulate Schwann cells to divide, demonstrating the specificity o f the membrane-bound mitogenic signal 1~. We report here that axolemma-enriched fractions isolated from adult rat and bovine peripheral nervous system (PNS) and central nervous system (CNS) contain a potent stimulus for mitosis o f Schwann cells cultured from rat dorsal root ganglion. Preparation of adequate material for detailed biochemical analysis o f the mitogcn should now be feasible. Axolemma-enriched fractions were isolated from rat sciatic nerve, bovine intradural root, bovine corpus callosum and crudely dissected rat brain stem. The procedures used for the isolation of these fractions and their biochemical and morphological characteristics have been recently reviewed 5. Briefly, a preparation of 0165-3806/82/0000-0000/$02.75 ,~ Elsevier Biomedical Press
296 m y e l i n a t e d axons was o b t a i n e d from thc dissociated PNS nerve tibers or C N S white matter by flotation in a buffered s a l t - s u c r o s e medium. Myelinated axons were disrupted by o s m o t i c a n d mechanical shock and an a x o l e m m a - c n r i c h e d fraction was isolated by a d i s c o n t i n u o u s density gradient centrifugation. PNS and CN S m e m b r a n e fractions were collected at the interface o f 1.0 M and 1.2 M suclose. This m e m b r a n e p r e p a r a t i o n is enriched in specific activity o f surface m e m b r a n c m a r k e r enzymes, d e m o n s t r a t e s specific binding o f t e t r o d o t o x i n (a sodium channel marker), and does not contain a p p r e c i a b l e a m o u n t s o f myelin basic p~otein or non-axolemm:~l m a r k e r enzymes .~,.The m e t h o d for the p r e p a r a t i o n o f pure, stable, non-dividing Schxs.ann cells has been described H. Purified m e m b r a n e fractions were in a d i a t e d with ultraviolet light for at least 15 min, and then a d d e d to the Schwann cell culture (5 -10 tzg protein per culture dish). After 24 h, fractions were again a d d e d to the culture, a~ was [ZH]thymidinc ( I t;Ci,ml), a n d incubation was c o n t i n u e d for an a d d i t i o n a l 24 h. The cultures were then fi.xed and processed for a u t o r a d i o g r a p h y I i. A labeling index ( n u m b e r o f labeled cell nuclei.'total cells ~,: 100) was d e t e r m i n e d by light microscopic observations on whole mounts o f these p r e p a r a t i o n s ~. As shown in Table 1, a x o l e m m a - e n r i c h e d fractions gave rise to a labeling index which was 20- to 40-fold elevated over that o f controls (Schwann cells to which no m e m b r a n e was added). It is evident f r o m Table I t h a t : (a) the Schwann cell can be stimulated to proliferate by m e m b r a n e fractions derived from both P N S and C N S o f a given species; (b) the mitogenic signal is not species restricted since rat Schv, ann cells can r e s p o n d equally well to rat or bovine a x o l e m m a - e n r i c h e d fractions: a n d (c) the TABLE I Potency of mitogenic signal in whole homogenate, axolemnm-enriched fi'action~ aml m)elin derived from rat attd bovine CNS and PNS myelinated axons
The labeling index represents the average proportion of Schwann cells labeled with [:q~]thymiditac from 2 fields of approximately 1000 cells (~: S.D.) when stimulated with 5-10 l,g protein per culture dish of each subcellular fraction tested. Subcellular ]?action tested
Control (no membrane) Axolemma-enriched fractions Rat CNS (25-day) Rat CNS (60-day) Bovine CNS (corpus collosum) Bovine PNS (intradural roots) Rat sciatic nerve Whole homogenate Axolemma.-enriched fraction* Myelin Rat CNS Whole homogenate Axolemma-enriched fraction Myelin * Single field only.
Labeling index
0.28 r 0.14 12.18 10.39 8.82 9.58
T 1.81 :~ 0.37 -_. 0.07 : 0.86
0.23 :i 0.13 6.7 I 0.15 _i 0.05 12.88 :t 0.95 9.78 :i 0.78 4.51 :t 0.07
297 mitogenic signal does not appear to be developmentally restricted since axolemmaenriched fractions from fully differentiated CNS or PNS can still cause Schwann cell proliferation. In addition, the potency of the axolemmal mitogenic signal does not markedly change in CNS preparations obtained from 25- or 60-day-old rats. In the axolemma preparation obtained from the PNS, the specific activity of the mitogenic signal is enriched about 50-fold over that of whole homogenate; the myelin fraction appears to have no effect on Schwann cell proliferation. In contrast, in CNS preparations there is no comparable increase in the potency of the mitogenic signal of the axolemma-enriched fraction over that of whole homogenate; in addition, the rat CNS myelin fraction is mitogenic. Part of the difference in the potency of mitogenic signal in the whole homogenate of PNS and CNS may be related to the extent to which the mitogenic signal is exposed in whole homogenate. Because the brain stem contains more unmyelinated nerve fibers than the peripheral nerve trunk (and unmyelinated fibers are known to be mitogenic for Schwann cellsH), a greater amount of mitogen may be exposed in CNS whole homogenates than in those from PNS where the axonal mitogen may be shielded by fragments of myelin. CNS myelin preparations are known to be contaminated to a limited extent by axolemma 8 which could partially account for the high labeling index observed in the CNS myelin fraction. However, the possibility that myelin is responsible for the stimulation of Schwann cell proliferation can not be ruled out at present. In this regard, it is of interest that myelinating Schwann cells (in contrast to non-myelinating cells) have been shown to divide specifically during Wallerian degeneration, suggesting that a myelin degeneration product may stimulate Schwann cell division 11. The nature of the membrane bound mitogenic signal was investigated by subjecting the axolemma-enriched fractions to trypsin and heat treatment as outlined in Table 11, followed by evaluation of this effect on thymidine incorporation into Schwann cells. Considering that freeze fracture 4 and neurotoxin binding studies 6 indicate that the bulk of the membrane vesicles in these preparations have a right-sideout orientation, the marked lability of the mitogenic signal to trypsin suggests that the mitogenic signal may reside on the outer surface of the axonal membrane in situ. Heat and trypsin lability also suggest the mitogen may be a protein. Incubation of the axolemma-enriched fraction at 34 °C for 24 h did not diminish the intensity of the mitogenic signal whereas the neurite mitogen isolated from cultured cells lost over T A B L E I1
Effect oJheat and tr),psht on mitogenic signal in rat C N S axolemma-enriched fractions All fractions were evaluated after the indicated treatment at saturating levels of a x o l e m m a - e n r i c h e d fractions per culture dish.
Treatment
Labeling index (° o of control}
None 0.05 % trypsin, 60 min, 34 °C 80'C,10 rain 34 C, 25 h
100.0% 3.0 2_ 3 4.0 ± 2% 118.0-:- 4
298 807,i of its mitogenic activity under these conditions ~3. In addition, the neuritc mitogen was inactivated by freezing whereas the potency of the mitogen in the axolemma-enriched preparations was not affected by freezing. These differences in mitogen lability suggest that either the molecular nature of the mitogen from the two sources is different or that the mitogen in the axolemma-enriched fraction is in a more protected environment than the identical mitogen in the neuritc. Since the membrane fractions used in these studies have been extensively washed during the preparation procedure, it does not seem likely that the mitogenic factor under consideration is a soluble factor which is non-specifically associated with the membrane fragments. Several soluble factors have been reported to be mitogenic in Schwann cells prepared by an alternative methodg.t~L One interesting aspect of these studies is the observation that axolem ma isolated from a fully differentiated, myelinated nerve tiber is capable of stimulating Schwann cell proliferation. This observation may be relevant to the proliferation of Schwann cells observed during segmental demyelination 2 and suggests the intact mature but unensheathed axon may be mitogenic. The fact that the PNS myelinating cell can respond to CNS axolemma is not unexpected since there are examples of: (a) nerve fibers originating in the CNS which in part of their course arc ensheathed and myelinated by Schwann cells (as are the somatic motor axons): and (b) nerve libers entirely resident within the CNS which under exceptional conditions may be myelinated by Schwann cells~. Future work will be directed toward the purification of this mitogen found on the surface of the axonal membrane of myelinated axons or unmyelinated neufites. Because neurites raised in culture provide very limited amounts of mitogenic material. while the axolemma-enriched preparations provide greater amounts of starting material, attempts at purification and elucidation of the molecular nature of this particulate mitogen are now feasible. This work was supported by the National Multiple Sclerosis Society (Grant A117-B-2 to G.H.D. and RG 1118 to R.P.B.); grants from the National Institutes of Health (NS 10821, NS 15408 to G.H.D., and GM 18450, NS 09923 to R.P.B.h and the National Science Foundation (BNS-78-02754 to G.H.D., and BM-77-15972 to R.P.B.). J.L.S. was supported by Medical Scientist Training Grant TO-5-GM-02016. I Aguayo, A. J., Martin, J. P. and Bray, G. M., Effects of nerve growth factor antiserum on peripheral unmyelinated fibers, Acta Neuropath., 20 (1972) 288 298. 2 Asbury, A. K. and Johnson, P. C. In Pathology of Peripheral Nerve, W. B. Saunders, Philadelphia, 1978, p. 60. 3 Bussow, H., Schwann cell myelin ensheathing CNS axons ill the nerve fiber layer of the cat retina, J. Neurocytol., 7 (1978) 207-214. 4 Cullen, M., DeVries, G. and Webster, H., Freeze fracture of isolated myelin and axolemma membrane fractions, Brain Res., 229 (1981) 311-322. 5 DeVries, G. H., Isolation of axolemma-enriched fractions from mammalian CNS. In N. Marks and R. Rodnight (Eds.), Methods in Neurochemistry, Vol. 5, Plenum Press, New York, 1981, pp. 3-38. 6 DeVries, G. H. and Lazdunski, M., Binding of neurotoxins to rat CNS axolemma-enriched fractions, Trans. Amer. Sot'. Neurochem., 12 (1981) 157.
299 7 McCarthy, K. D. and Partlow, L. M., Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells, Brain Res., 114 (1976) 415--426. 8 Mclllwain, D. L., Localization of the acetylcholinesterase-containing membranes in purified myelin fractions, Brain Res., 69 (1974) 182-187. 9 Raft, M. C., Abney, E., Brockes, J. P. and Hornby-Smith, A., Schwann cell growth factors, Cell, 15 (1978) 813-822. 10 Raft, M. C., Hornby-Smith, A. and Brockes, J. P., Cyclic AMP as a mitogenic signal for cultured rat Schwann cells, Nature (Lond.), 273 (1978) 672-673. 11 Salzer, J. L. and Bunge, R. P., Studies of Schwann cell proliferation: I. An analysis in tissue culture of proliferation during development, Wallerian degeneration, and direct injury, J. Cell Biol., 84 (1980) 739-752. 12 Salzer, J. L., Bunge, R. P. and Glaser, L., Studies of Schwann cell proliferation: 111. Evidence for the surface localization of the neurite mitogen, J. Cell Biol., 84 (1980) 767-778. 13 Salzer, J. L., Williams, A. K., Glaser, L. and Bunge, R. P., Studies of Schwann cell proliferation : II. Characterization of the stimulation and specificity of the response to a neurite membrane fraction, J. Cell Biol., 84 (1980) 753-766. 14 Wood, P. M., Separation of functional Schwann cells and neurons from normal peripheral nerve tissue, Brahl Res., 115 (1976) 361-375. 15 Wood, P. M. and Bunge, R. P., Evidence that sensory axons are mitogenic for Schwann cells, Nature (Lond./, 256 (1975) 662-664.
295
Elsevier Biomedical Press
Short Communications
Axolemma-enriched fractions isolated from PNS and CNS are mitogenic for cultured Schwann cells
GEORGE H. DeVRIES, JAMES L. SALZER and RICHARD P. BUNGE
Department of Biochemistry, Medical College of Virginia, Richmond, VA 23298 and (J.L.S. anti R.P.B.) Departments of Anatomy and Neurobioh~gy, Washington University School of Medicine, St. Louis, MO 63110 I U.S.A.)
(Accepted October 12th, 1981) Key words: axolemma fractions - - cultured Schwann cells -- mitogenesis -- myelinated axons
Axolemma-enriched fractions isolated from myelinated axons of rat and bovine CNS and PNS will stimulate a qmescent population of cultured rat Schwann cells to proliferate, The mitogen in the axolemma-enriched fraction is not lost in the extensive washing used to isolate the membrane fraction and is sensitive to heat and trypsin, suggesting that it is an integral membrane protein or glycoprotein. The axolemma-enriched preparations should provide an adequate source of Schwann cell mitogen for solubilization and purification of this membrane bound mitogen. Schwann cell proliferation appears to be regulated by axonal contact. This is suggested by both the in vitro observation that Schwann cell proliferation is dependent on a mitogenic signal provided by growing neurons7, t~, and the in vivo observation that Schwann cell numbers in nerve trunks are proportional to the number o f nerve fibers present t. Tissue culture preparations utilizing separated, pure populations o f sensory neurons and Schwann cells tt demonstrate that a mitogenic signal can be provided to Schwann cells by intact bare sensory axons11, t~, that cell-cell contact is required 12, and that a microsomal fraction derived from cultured neurites can substitute for the intact axon 13. Membrane fractions derived from other cell types in tissue culture did not stimulate Schwann cells to divide, demonstrating the specificity o f the membrane-bound mitogenic signal 1~. We report here that axolemma-enriched fractions isolated from adult rat and bovine peripheral nervous system (PNS) and central nervous system (CNS) contain a potent stimulus for mitosis o f Schwann cells cultured from rat dorsal root ganglion. Preparation of adequate material for detailed biochemical analysis o f the mitogcn should now be feasible. Axolemma-enriched fractions were isolated from rat sciatic nerve, bovine intradural root, bovine corpus callosum and crudely dissected rat brain stem. The procedures used for the isolation of these fractions and their biochemical and morphological characteristics have been recently reviewed 5. Briefly, a preparation of 0165-3806/82/0000-0000/$02.75 ,~ Elsevier Biomedical Press
296 m y e l i n a t e d axons was o b t a i n e d from thc dissociated PNS nerve tibers or C N S white matter by flotation in a buffered s a l t - s u c r o s e medium. Myelinated axons were disrupted by o s m o t i c a n d mechanical shock and an a x o l e m m a - c n r i c h e d fraction was isolated by a d i s c o n t i n u o u s density gradient centrifugation. PNS and CN S m e m b r a n e fractions were collected at the interface o f 1.0 M and 1.2 M suclose. This m e m b r a n e p r e p a r a t i o n is enriched in specific activity o f surface m e m b r a n c m a r k e r enzymes, d e m o n s t r a t e s specific binding o f t e t r o d o t o x i n (a sodium channel marker), and does not contain a p p r e c i a b l e a m o u n t s o f myelin basic p~otein or non-axolemm:~l m a r k e r enzymes .~,.The m e t h o d for the p r e p a r a t i o n o f pure, stable, non-dividing Schxs.ann cells has been described H. Purified m e m b r a n e fractions were in a d i a t e d with ultraviolet light for at least 15 min, and then a d d e d to the Schwann cell culture (5 -10 tzg protein per culture dish). After 24 h, fractions were again a d d e d to the culture, a~ was [ZH]thymidinc ( I t;Ci,ml), a n d incubation was c o n t i n u e d for an a d d i t i o n a l 24 h. The cultures were then fi.xed and processed for a u t o r a d i o g r a p h y I i. A labeling index ( n u m b e r o f labeled cell nuclei.'total cells ~,: 100) was d e t e r m i n e d by light microscopic observations on whole mounts o f these p r e p a r a t i o n s ~. As shown in Table 1, a x o l e m m a - e n r i c h e d fractions gave rise to a labeling index which was 20- to 40-fold elevated over that o f controls (Schwann cells to which no m e m b r a n e was added). It is evident f r o m Table I t h a t : (a) the Schwann cell can be stimulated to proliferate by m e m b r a n e fractions derived from both P N S and C N S o f a given species; (b) the mitogenic signal is not species restricted since rat Schv, ann cells can r e s p o n d equally well to rat or bovine a x o l e m m a - e n r i c h e d fractions: a n d (c) the TABLE I Potency of mitogenic signal in whole homogenate, axolemnm-enriched fi'action~ aml m)elin derived from rat attd bovine CNS and PNS myelinated axons
The labeling index represents the average proportion of Schwann cells labeled with [:q~]thymiditac from 2 fields of approximately 1000 cells (~: S.D.) when stimulated with 5-10 l,g protein per culture dish of each subcellular fraction tested. Subcellular ]?action tested
Control (no membrane) Axolemma-enriched fractions Rat CNS (25-day) Rat CNS (60-day) Bovine CNS (corpus collosum) Bovine PNS (intradural roots) Rat sciatic nerve Whole homogenate Axolemma.-enriched fraction* Myelin Rat CNS Whole homogenate Axolemma-enriched fraction Myelin * Single field only.
Labeling index
0.28 r 0.14 12.18 10.39 8.82 9.58
T 1.81 :~ 0.37 -_. 0.07 : 0.86
0.23 :i 0.13 6.7 I 0.15 _i 0.05 12.88 :t 0.95 9.78 :i 0.78 4.51 :t 0.07
297 mitogenic signal does not appear to be developmentally restricted since axolemmaenriched fractions from fully differentiated CNS or PNS can still cause Schwann cell proliferation. In addition, the potency of the axolemmal mitogenic signal does not markedly change in CNS preparations obtained from 25- or 60-day-old rats. In the axolemma preparation obtained from the PNS, the specific activity of the mitogenic signal is enriched about 50-fold over that of whole homogenate; the myelin fraction appears to have no effect on Schwann cell proliferation. In contrast, in CNS preparations there is no comparable increase in the potency of the mitogenic signal of the axolemma-enriched fraction over that of whole homogenate; in addition, the rat CNS myelin fraction is mitogenic. Part of the difference in the potency of mitogenic signal in the whole homogenate of PNS and CNS may be related to the extent to which the mitogenic signal is exposed in whole homogenate. Because the brain stem contains more unmyelinated nerve fibers than the peripheral nerve trunk (and unmyelinated fibers are known to be mitogenic for Schwann cellsH), a greater amount of mitogen may be exposed in CNS whole homogenates than in those from PNS where the axonal mitogen may be shielded by fragments of myelin. CNS myelin preparations are known to be contaminated to a limited extent by axolemma 8 which could partially account for the high labeling index observed in the CNS myelin fraction. However, the possibility that myelin is responsible for the stimulation of Schwann cell proliferation can not be ruled out at present. In this regard, it is of interest that myelinating Schwann cells (in contrast to non-myelinating cells) have been shown to divide specifically during Wallerian degeneration, suggesting that a myelin degeneration product may stimulate Schwann cell division 11. The nature of the membrane bound mitogenic signal was investigated by subjecting the axolemma-enriched fractions to trypsin and heat treatment as outlined in Table 11, followed by evaluation of this effect on thymidine incorporation into Schwann cells. Considering that freeze fracture 4 and neurotoxin binding studies 6 indicate that the bulk of the membrane vesicles in these preparations have a right-sideout orientation, the marked lability of the mitogenic signal to trypsin suggests that the mitogenic signal may reside on the outer surface of the axonal membrane in situ. Heat and trypsin lability also suggest the mitogen may be a protein. Incubation of the axolemma-enriched fraction at 34 °C for 24 h did not diminish the intensity of the mitogenic signal whereas the neurite mitogen isolated from cultured cells lost over T A B L E I1
Effect oJheat and tr),psht on mitogenic signal in rat C N S axolemma-enriched fractions All fractions were evaluated after the indicated treatment at saturating levels of a x o l e m m a - e n r i c h e d fractions per culture dish.
Treatment
Labeling index (° o of control}
None 0.05 % trypsin, 60 min, 34 °C 80'C,10 rain 34 C, 25 h
100.0% 3.0 2_ 3 4.0 ± 2% 118.0-:- 4
298 807,i of its mitogenic activity under these conditions ~3. In addition, the neuritc mitogen was inactivated by freezing whereas the potency of the mitogen in the axolemma-enriched preparations was not affected by freezing. These differences in mitogen lability suggest that either the molecular nature of the mitogen from the two sources is different or that the mitogen in the axolemma-enriched fraction is in a more protected environment than the identical mitogen in the neuritc. Since the membrane fractions used in these studies have been extensively washed during the preparation procedure, it does not seem likely that the mitogenic factor under consideration is a soluble factor which is non-specifically associated with the membrane fragments. Several soluble factors have been reported to be mitogenic in Schwann cells prepared by an alternative methodg.t~L One interesting aspect of these studies is the observation that axolem ma isolated from a fully differentiated, myelinated nerve tiber is capable of stimulating Schwann cell proliferation. This observation may be relevant to the proliferation of Schwann cells observed during segmental demyelination 2 and suggests the intact mature but unensheathed axon may be mitogenic. The fact that the PNS myelinating cell can respond to CNS axolemma is not unexpected since there are examples of: (a) nerve fibers originating in the CNS which in part of their course arc ensheathed and myelinated by Schwann cells (as are the somatic motor axons): and (b) nerve libers entirely resident within the CNS which under exceptional conditions may be myelinated by Schwann cells~. Future work will be directed toward the purification of this mitogen found on the surface of the axonal membrane of myelinated axons or unmyelinated neufites. Because neurites raised in culture provide very limited amounts of mitogenic material. while the axolemma-enriched preparations provide greater amounts of starting material, attempts at purification and elucidation of the molecular nature of this particulate mitogen are now feasible. This work was supported by the National Multiple Sclerosis Society (Grant A117-B-2 to G.H.D. and RG 1118 to R.P.B.); grants from the National Institutes of Health (NS 10821, NS 15408 to G.H.D., and GM 18450, NS 09923 to R.P.B.h and the National Science Foundation (BNS-78-02754 to G.H.D., and BM-77-15972 to R.P.B.). J.L.S. was supported by Medical Scientist Training Grant TO-5-GM-02016. I Aguayo, A. J., Martin, J. P. and Bray, G. M., Effects of nerve growth factor antiserum on peripheral unmyelinated fibers, Acta Neuropath., 20 (1972) 288 298. 2 Asbury, A. K. and Johnson, P. C. In Pathology of Peripheral Nerve, W. B. Saunders, Philadelphia, 1978, p. 60. 3 Bussow, H., Schwann cell myelin ensheathing CNS axons ill the nerve fiber layer of the cat retina, J. Neurocytol., 7 (1978) 207-214. 4 Cullen, M., DeVries, G. and Webster, H., Freeze fracture of isolated myelin and axolemma membrane fractions, Brain Res., 229 (1981) 311-322. 5 DeVries, G. H., Isolation of axolemma-enriched fractions from mammalian CNS. In N. Marks and R. Rodnight (Eds.), Methods in Neurochemistry, Vol. 5, Plenum Press, New York, 1981, pp. 3-38. 6 DeVries, G. H. and Lazdunski, M., Binding of neurotoxins to rat CNS axolemma-enriched fractions, Trans. Amer. Sot'. Neurochem., 12 (1981) 157.
299 7 McCarthy, K. D. and Partlow, L. M., Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells, Brain Res., 114 (1976) 415--426. 8 Mclllwain, D. L., Localization of the acetylcholinesterase-containing membranes in purified myelin fractions, Brain Res., 69 (1974) 182-187. 9 Raft, M. C., Abney, E., Brockes, J. P. and Hornby-Smith, A., Schwann cell growth factors, Cell, 15 (1978) 813-822. 10 Raft, M. C., Hornby-Smith, A. and Brockes, J. P., Cyclic AMP as a mitogenic signal for cultured rat Schwann cells, Nature (Lond.), 273 (1978) 672-673. 11 Salzer, J. L. and Bunge, R. P., Studies of Schwann cell proliferation: I. An analysis in tissue culture of proliferation during development, Wallerian degeneration, and direct injury, J. Cell Biol., 84 (1980) 739-752. 12 Salzer, J. L., Bunge, R. P. and Glaser, L., Studies of Schwann cell proliferation: 111. Evidence for the surface localization of the neurite mitogen, J. Cell Biol., 84 (1980) 767-778. 13 Salzer, J. L., Williams, A. K., Glaser, L. and Bunge, R. P., Studies of Schwann cell proliferation : II. Characterization of the stimulation and specificity of the response to a neurite membrane fraction, J. Cell Biol., 84 (1980) 753-766. 14 Wood, P. M., Separation of functional Schwann cells and neurons from normal peripheral nerve tissue, Brahl Res., 115 (1976) 361-375. 15 Wood, P. M. and Bunge, R. P., Evidence that sensory axons are mitogenic for Schwann cells, Nature (Lond./, 256 (1975) 662-664.