- Email: [email protected]
Effects of nerve growth factor and heart cell conditioned medium on neurite regeneration of aged sympathetic neurons in culture
Brain Research, 348 (1985) 100-106
100
Elsevier BRE 11145
Effects of Nerve Growth Factor and Heart Cell Conditioned Medium on Neurite Regeneration of Aged Sympathetic Neurons in Culture YOKO UCHIDA and MASANORI TOMONAGA
Department of Clinical Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2, Sakae-cho, Itabashi-ku, Tokyo-173 (Japan) (Accepted February 5th, 1985)
Key words: nerve growth factor - - heart-cell-conditioned medium - - aging - - sympathetic neuron - neurite regeneration - - cell culture
The effects of nerve growth factor (NGF) and heart-cell-conditioned medium (HCM) on the neurite regeneration of aged sympathetic neurons were investigated in culture. Investigation of HCM was carried out by two different methods: one was the use of whole HCM on collagen substratum, which reflected component(s) effective in solution (HCM-S); the other was the use of polyornithine (PORN)-binding component(s) (P-HCM). Superior cervical ganglion neurons prepared from male mice from 6 to 30 months of age were cultured in MEM-10% FCS on collagen or gelatin-PORN substratum for 3 days. The number of neurons with neurites and the length of neurites were quantified as neurite production and elongation, respectively. Neuronal survival was not affected by addition of NGF, HCM-S or P-HCM. Neurite production of early adult neurons was enhanced by NGF, HCM-S or P-HCM. In contrast, neurite production of aged neurons was enhanced by only HCM-S, but not NGF or P-HCM. HCM-S did not promote neurite elongation in neurons at any age. Neurite elongation of early adult neurons was enhanced by NGF or P-HCM. Neurite elongation of aged neurons was enhanced by P-HCM. However, responsiveness of NGF for neurite elongation varied according to substrata. No age-related difference was found in neurite production and elongation in the absence of NGF, HCM-S or P-HCM. These results indicate that responsiveness of aged sympathetic neurons is various in different growth factors.
INTRODUCTION
ability of neurons to respond to growth factors. In a previous study 30, we showed that responsive-
Since the n u m b e r of axons and synapses decrease in aged animals, the regenerative process may be damaged in the aged nervous system. In fact, several studies indicate that axonal sprouting and synaptic formation in aged nervous system decline after partial denervation in viv08,13,23.2s. As an explanation for
ness to N G F was diminished in sympathetic ganglion neurons from aged mice in explant culture. In ganglion explants, however, many n o n - n e u r o n a l cells as
the diminished plasticity in aged animals, the following speculation has been presented28: the aged neurons may have less ability to synthesize or assemble materials necessary for growth; growth-inducing substances may not be readily available; or the target cells may be less able to accept new innervation. In vivo studies have not given a satisfactory explanation of the mechanism of diminished plasticity in aged neurons. Tissue or cell culture technique is useful for understanding these mechanisms4, ~8. However, the reason for this reduction in plasticity is not k n o w n yet. It is, therefore, of interest to determine whether this diminished plasticity is due to a reduction in the
well as neurons exist and release several substances into medium. Therefore, the regenerative response of ganglion explants is possibly affected by not only N G F , but also other factors which are released from explants themselves2, 21. Besides chemically defined substances, such as N G F , fibronectin27 and laminin27, several materials which promote n e u r o n a l survival or neurite outgrowth of embryonic sympathetic neurons have been found in various tissue extracts and conditioned media1,11,12A4A7,32. In some cases, the neurite outgrowth-promoting activity has been shown to bind directly to the polycation substratum and operate only in its substratum-associated condition1,11. In the present study, we examined whether dissociated aged sympathetic n e u r o n s respond to N G F
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B .V. (Biomedical Division)
101 and heart-cell-conditioned medium (HCM) in culture. With regard to the latter, we tested the two different components of HCM: one is effective in solution32, the other is effective in polyornithine (PORN)-binding conditionl,~l. MATERIALS AND METHODS Media and reagents Media and reagents used were: CMF, Ca 2+, Mg 2+free Hank's balanced salt solution; medium I, 90% Eagle's minimal essential medium (MEM), 10% fetal calf serum (FCS, Gibco), 6 mg/ml glucose, 5 pg/ml gentamycin, 10-5 M cytosine arabinoside (Ara-C) and 10-5 M fluorodeoxyuridine (FdU)33; medium II: this was identical to medium I except that Ara-C and FdU were deleted33; NGF, 2.5S nerve growth factor (Collaborative Research); NGF antiserum, rabbit antiserum against 2.5S NGF (Collaborative Research); Heart-cell-conditioned medium (HCM), heart cells were dissociated from 9 days chick embryos, and grown to confluent in M E M - 1 0 % FCS. HCM was obtained by exposing the culture to fresh MEM-10% FCS for 48 h 31.
ture plates precoated with collagen or gelatin-PORN and fed in medium I for 24 h, and then in medium II for a further 48 h (37 °C, 5% CO2-air). To determine the effect of NGF, 10 or 100 ng/ml of NGF was added to medium I and medium II. To test the effect of HCM component(s) which is effective in solution, SCG neurons were fed on collagen substratum in medium I and II, containing HCM (0.5 ml/ml) and NGF antiserum (2.5/A/ml). To determine the effect of PORN-binding component(s) of HCM, gelatinPORN precoated wells were incubated with 1 ml/well of either HCM or M E M - 1 0 % FCS (control) for 24 h at 37 °C in 5% CO2-airk After the medium was removed, wells were washed with fresh M E M - 1 0 % FCS and seeded with SCG cells. The culture was fed in medium I and II. After 3 days in culture, cells were fixed with 2% glutaraldehyde in MEM. All single neurons and the single neurons with neurites at least twice the cell diameter were counted. The neurite length was determined by measuring the distance between the end of the longest neurite and the cell body. Measurement was done for 10 neurons in each well. RESULTS
Substrata Collagen prepared from rat tail tendon9 was spread on culture plates (containing 24 mm diameter wells), and gelled by exposure to NH 3 vaper for 5 min. The culture plates were then washed extensively with MEM. Gelatin-polyornithine (PORN)-coated plates were prepared according to the procedure previously reported 22 except that PORN concentration was reduced to 0.1 mg/ml.
The living cell populations at the time of isolation were 72 _+ 5%, 73 _+ 5% and 70 _+ 5% from mice 6, 18 and 24-30 months of age, respectively. No difference was detected in living cell population among the 3 groups.
100
Neuronal cell culture Superior cervical ganglia (SCG) were dissected from male C57BL/6 mice 6, 18 and 24-30 months of age. Decapsulated ganglia were treated with CMF containing 0.25% trypsin-collagenase at 37 °C for 15 rain. At the end of the incubation, M E M - 1 0 % FCS was added to stop digestion. Then ganglia were re-digested with 0.25% collagenase in CMF at 37 °C for 2 h. Dissociation was carried out in M E M - 1 0 % FCS by gentle aspiration using a heat-polished, glass Pasteur pipette. SCG neurons (1600 cells/well) were seeded on cul-
A
[] 0 ng/m[ [ ] 'tO nglml • 100 ng/ml
B
g
"650 i 6
18
24-30 6 Months of age
18
24-30
Fig. 1. Effect of NGF on survival of SCG neurons from mice 61 18 and 24-30 months of age at 3 days in culture on the collagen (A) or gelatin-PORN (B). Each value represents the mean + S.E.M. of 5 determinations.
102 Neuronal response to N G F As Fig. 1 shows, neuronal survival at 3 days in culture on the collagen was slightly higher than that on the gelatin-PORN substratum, but not significantly different (P > 0.1). Neuronal survival was not affected by either the age of the neuron or addition of N G F (Fig. 1). The neurite production, expressed as a percentage of neurons with neurites, on gelatin-PORN was significantly higher (about 3-fold) than that on the collagen substratum in all age groups with or without of N G F (Fig. 2). As shown in Fig. 2, no difference was found in neurite production among 3 groups of neurons cultured without N G F on either substratum. N G F enhanced the neurite production of 6-month neurons, but not of neurons 18 months or more on collagen or gelatin-PORN substratum (Fig. 2). As Fig. 3 shows, the neurite elongation, expressed as neurite length, of neurons of 18-months or more, was almost the same as that of 6-month neurons in the absence of N G F on either substratum. Whereas neurite elongation of 6-month neurons was enhanced by addition of N G F on either substratum, it was somewhat different in aged neurons. Neurite elongation of neurons of 18-months or more was enhanced by N G F on the collagen (Fig. 3A), but not on the gelatin-PORN (Fig. 3B). Neuronal response to H C M component(s) in solution (HCM-S) As shown in Fig. 4A, HCM-S did not increase the
[] []
3O
0 ng/ml 10 ng/ml
==
.,ooze,m,
t2O
.o
B
i ll
A
[] [] •
v
B
0 ng/mt 10 nglrnl 100 ng/ml
5OO
250 o) Z
0 18
6
24-30
6
18
24-30
Months of ccje
Fig. 3. Effect of N G F on neurite length of SCG neurons from mice 6, 18 and 2 4 - 3 0 m o n t h s of age at 3 days in culture on the collagen (A) or gelatin-PORN (B). Each value represents the mean _+ S.E.M. of 5 determinations. Significant differences from control (NGF 0 ng/ml) at P < 0.05 (*) and P < 0.005
(***).
number of surviving neurons in any age group. The neurite production in all age groups was enhanced in the presence of HCM-S (Fig. 4B). This component did not enhance the neurite elongation of early adult or aged neurons (Fig. 4C).
[] []
6O
"6
Control HCM
30
0
c c
5~
2°f ~o 0
400
~o -~
"6
200
0 6
18
24-30
6
18
2/.-30
Months of age
Fig. 2. Effect of N G F on percentage of SCG neurons with neurite. Neurons from mice 6, 18 and 24-30 m o n t h s of age were cultured on the collagen (A) or gelatin-PORN (B) for 3 days. Each value represents the m e a n + S.E.M. of 5 determinations. Significant differences from control ( N G F 0 ng/ml) at P < 0.05 (*) and P < 0.025 (**) estimated using Student's t-test.
6
18
24-30
Months of acje
Fig. 4. Effects of H C M on survival (A), percentage of neurons with neurite (B) and the length of neurite (C) of SCG neurons of various ages at 3 days in culture on the collagen. Each value represents the m e a n _+ S.E.M. of 5 determinations. Significant differences from control at P < 0.05 (*) and P < 0.005 (***).
103
Neuronal response to PORN-binding component(s) of HCM (P-HCM) As Fig. 5A shows, P - H C M did not increase the neuronal survival in any age group with or without NGF. The neurite production was enhanced by PH C M in 6-month neurons, but not in neurons of 18months or more (Fig. 5B). The combination of PH C M and N G F increased the neurite production of both 6- and 18-month neurons (Fig. 5B). Nevertheless, aged neurons (24-30 months of age) did not increase their neurite production in the presence of both P - H C M and N G F (Fig. 5B). On the other hand, the neurite elongation was enhanced drastically by the addition of P - H C M in all age groups (Figs. 5C and 6). The combination of P - H C M and N G F also enhanced the neurite elongation of all age groups (Fig. 5C). DISCUSSION The results indicate that: (1) neuronal survival is not enhanced by either N G F or H C M in any age groups; (2) the responsiveness to N G F for both neurite production and elongation on P O R N substratum declines in aged neurons; (3) H C M component(s) in solution enhances the neurite production but not the elongation in any age groups; (4) responsiveness of neurons to PORN-binding component(s) of H C M for
A
[] [] []
6C
Controt ~-HCM F-HCM ÷ NGF
J
20
13 -
30
c .c
~c
2o
o
2
-
I
10
c
6
18
M o n t h s of a g e
24-30
6
18
1
24-30
M o n t h s of a g e
Fig. 5. Effects of PORN-binding fraction of HCM on survival (A), percentage of neurons with neurite (B), and neurite length (C) of SCG neurons from various age mice at 3 days in culture. Each value represents the mean + S.E.M. of 5 determinations. Significant differences from control at P < 0.05 (*), P < 0.025 (**) and P < 0.005 (***).
neurite production but not neurite elongation, decreases with age. The second result is somewhat different from our previous observation30 that neurite elongation of SCG neurons from mice 24 months of age was enhanced by a high concentration of N G F in explant culture. This discrepancy may be because of the difference in culture method (explant culture vs dissociation culture). In explant culture, high concentration of N G F may promote the release of some kind of neurite-promoting substances into media from numerous neuronal and non-neuronal cells. In fact, ganglion itself produces substances that act as neurite promoting factors by binding to the substrate surrounding the ganglion2, 21. In dissociation culture, because of low cell density (1600 cells/24 m m well), little substances may be released. Other possible causes are that the aged neurons might be damaged more severely by enzymatic dissociation, or be less capable of recovery from injury. Trypsin is reported to inactivate N G F receptor of the m e m b r a n e fraction of SCG 5. However, it should be noted that: (1) the survival of aged neurons was almost the same as that of early adult neurons; (2) even aged neurons have the ability to respond to N G F for neurite elongation on collagen substratum. Our observation also indicates the following influences of substratum on the neurite regeneration: (1) gelatin-PORN supports more extensive neurite regeneration than collagen does with or without N G F regardless of neuronal age; and (2) neurite elongation of aged neurons is enhanced by N G F on the collagen but not on the gelatin-PORN. The former conclusion is consistent with an earlier study by Hawrot 22. The latter difference, based on substratum, cannot be explained at present. It might reflect that the promotion of neurite elongation by N G F was affected by cell surface materials involved in cellP O R N interaction 10, and the synthesis of these materials was suppressed in aged neurons. N G F is a protein essential for survival, development and differentiation of embryonic peripheral sympathetic neurons both in vivo and in vitro. Moreover, N G F plays a role in maintenance of adrenergic function in adult sympathetic neurons 3,7,19,20,26. However, the issue of whether adult sympathetic neurons depend on N G F for survival in vivo is not consistent 19,20,26. In culture of sympathetic neurons, N G F
104
Fig. 6. Phase-contrast micrographs of SCG neurons from mice 6 (a, c, e) and 30 (b, d, f) months of age, cultured for 3 days in the absence (a, b, e, f) and presence (c, d) of NGF. The gelatin-PORN substratum was pretreated before cell seeding with either fresh MEM-FCS medium (a, b, c, d) or heart-cell conditioned medium (e, f).
105 becomes less and less required for cell survival at later embryonic stages 29. O u r observations agree with this conclusion that N G F - d e p e n d e n c y for survival decreases in adult neurons. N G F is r e p o r t e d to exert a stimulatory effect on the regrowth of adrenergic fibers in the young adult (less than 3 months of age) after 6 - O H D A lesioning in vivo6. H o w e v e r , little is known about the action of N G F on neurite regeneration in adult or aged neurons. W e provided the first evidence that: (1) N G F p r o m o t e s both neurite production and elongation in adult neurons; but (2) the action of N G F for neurite production decreases with aging. It remains to be det e r m i n e d whether decreased responsiveness to N G F in aged neurons is due to the alteration in r e c e p t o r site or intercellular mechanisms which lead to neurite regeneration. Recently, several kinds of n e u r o n o t r o p h i c or neurite-promoting factors for peripheral ganglionic neurons are found in various tissue extracts and cell-conditioned media. Some of them are b l o c k e d by antiserum against NGF15, 24, but others are effective, even in the presence of anti-NGFl,ll,12,14,17,32. Since none of the factors are chemically defined, some questions arise: (1) is H C M - S actually N G F itself?; and (2) are H C M - S and P - H C M used in this study identical to the substances previously reportedkll,32? It is unlikely that H C M - S is itself N G F , because antiserum against mouse 2.5S N G F was a d d e d to block the activity of N G F in H C M . It should be stressed that species difference was not critical element, since antiserum to mouse 2.5S N G F effectively blocks the N G F - l i k e activity in the chick irides 16. Moreover, the effect of H C M - S on adult neurons is
REFERENCES 1 Adler, R., Manthorpe, M., Skaper, S.D. and Varon, S., Polyornithine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons, Brain Research, 206 (1981) 129-144. 2 Adler, R. and Varon, S., Neuritic guidance by polyornithine-attached materials of ganglionic origin, Dev. Biol., 81 (1981) 1-11. 3 Angeletti, P.V., Levi-Montalcini, R. and Caramia, F., Analysis of the effect of the antiserum to nerve growth factor in adult mice, Brain Research, 27 (1971) 343-355. 4 Argiro, V. and Johnson, M.I., Patterns and kinetics of neurite extension from sympathetic neurons in culture are age dependent, J. Neurosci., 2 (1982) 503-512.
not different from that of N G F , namely, N G F but not H C M - S p r o m o t e s the neurite elongation. The factors in H C M - S and P - H C M may correspond to the factors r e p o r t e d by V a r o n et al. 32 and A d l e r et al.1., respectively, because H C M - S and PH C M were p r e p a r e d from the same source by the same methods as the)' used. On the other hand, they used embryonic ganglion neurons, whereas we used adult neurons to examine the effect of trophic factors. The possibility could not be ruled out that the factors in H C M which we used were different from those previously r e p o r t e d 1,11,32, because the requirement of cultured sympathetic neurons for trophic factors changes during d e v e l o p m e n t 17. The isolation and characterization of H C M - S and P - H C M should provide the information on the similarity or dissimilarity among those factors. In conclusion, adult mouse sympathetic neurons kept in culture respond to N G F or H C M factors for neurite regeneration. H o w e v e r , in the aged neurons, the responsiveness to these factors varies between different growth factors, or different aspects of regeneration, such as neurite production or elongation. In other words, some, mechanisms for neurite regeneration may be disordered, but others may normally act in aged neurons. ACKNOWLEDGEMENTS This work was s u p p o r t e d in part by the Institute of Physical and Chemical Research as a part of the Project on Discovery of factors Regulating Aging. W e wish to thank Dr. F. Mizobe for his helpful discussions.
5 Banerjee, S.P., Cautrecasas, P. and Snyder, S.H., Nerve growth factor receptor binding, J. Biol. Chem., 250 (1975) 1427-1433. 6 Bjerre, B., Bjorklund, A. and MoNey, W., A stimulatory effect by nerve growth factor on the regrowth of adrenergic nerve fibers in the mouse peripheral tissues after chemical sympathectomy with 6-hydroxydopamine, Z. Zellforsch., 146 (1973) 15-43. 7 Bjerre, B., Wiklung, L. and Edwards, D.C., A study of the de- and regenerative changes in the sympathetic nervous system of the adult mouse after treatment with antiserum to nerve growth factor, Brain Research, 92 (1975) 257-278. 8 Black, M.M. and Lasek, R.J., Slowing of the rate of axonal regeneration during growth and maturation, Exp. Neurol., 63 (1979) 108-119.
106 9 Bornstein, M.B., Reconstituted rat-tail collagen used as substrate for tissue cultures on coverslips in maximow slides and roller tubes, Lab. Invest., 7 (1958) 134-137. 10 Chernoff, E.A.G., Maresh, G.A. and Culp, L.A., Isolation and characterization of a large, neurite-associated glycoconjugate from neuroblastoma cells, J. Cell Biol., 96 (1983) 661-668. 11 Collins, F., Induction of neurite outgrowth by a conditioned-medium factor bound to culture substratum, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 5210-5213. 12 Collins, F., Neurite outgrowth induced by the substrate associated material from nonneuronal cells, Dev. Biol., 79 (1980) 247-252. 13 Cotman, C.W. and Scheff, S.W., Compensatory synapse growth in aged animals after neuronal death, Mech. Aging Develop., 9 (1979) 103-117. 14 Coughlin, M.D., Bloom, E.M. and Black, I.B., Characterization of a neuronal growth factor from mouse heart-cellconditioned medium, Dev. Biol., 82 (1981) 56-68. 15 Crutcher, K.A. and Collins, F., In vitro evidence for two distinct hippocampal growth factors: basis of neuronal plasticity?, Science, 217 (1982) 67-68. 16 Ebendal, T., Hedlund, K.-O. and Norrgren, G., Nerve growth factors in chick tissues, J. Neurosci. Res., 8 (1982) 153-164. 17 Edgar, D., Barde, Y.-A. and Thoenen, H., Subpopulations of cultured chick sympathetic neurons differ in their requirements for suvival factors, Nature (London), 289 (1981) 294-295. 18 Fukuda, J. and Yamaguchi, K., Nerve cells of senescent mouse grown in tissue culture, Neurosci. Lett., 28 (1982) 331-335. 19 Goedert, M., Otten, U. and Thoenen, H., Biochemical effect of antibodies against nerve growth factor on developing and differentiated sympathetic ganglia, Brain Research, 148 (1978) 264-268. 20 Gorin, P.D. and Johnson, E.M., Effects of long-term nerve growth factor deprivation on the nervous system of the adult rat: an experimental autoimmune approach, Brain Research, 198 (1980) 27-42. 21 Gundersen, R.W. and Barrett, J.N., Neurite growth conesubstratum adherance increases in vitro, Dev. Brain Res., 12 (1984) 21-26.
22 Hawrot, E., Cultured sympathetic neurons: effects of cellderived and synthetic substrata o n survival and development, Dev. Biol., 74 (1980) 136-151. 23 Hulsebosch, C.E. and Coggeshall, R.E., Age related sprouting of dorsal root axons after sensory denervation, Brain Research, 288 (1983) 77-83. 24 Lindsay, R.M., Adult rat brain astrocytes support survival of both NGF-dependent and NGF-insensitive neurones, Nature (London), 282 (1979) 80-82. 25 McWilliams, J.R. and Lynch, G., Synaptic density and axonal sprouting in rat hippocampus: stability in adulthood and decline in late adulthood, Brain Research, 294 (1984) 152-156. 26 Otten, U., Goedert, M., Schwab, M. and Thibault, J., Immunization of adult rats against 2.5 S NGF: effects on the peripheral sympathetic nervous system, Brain Research, 176 (1979) 79-90. 27 Rogers, S.L., Letourneau, P.C., Palm, S.L., McGarthy, J. and Furcht, L.T., Neurite extension by peripheral and central nervous system neurons in response to substratumbound fibronectin and laminin, Dev. Biol., 98 (1983) 212-220. 28 Scheff, S.W., Bernardo, L.S. and Cotman, C.W., Decrease in adrenergic axon sprouting in the senescent rat, Science, 202 (1978) 775-778. 29 Selak, I., Skaper, S.D. and Varon, S., Ionic behavior and neuronal survival in developing ganglia: III. Studies with embryonic chick sympathetic neurons, J. Cell Physiol., 114 (1983) 229-234. 30 Uchida, Y. and Tomonaga, M., Nerve growth factor accelates regeneration of cultured adult sympathetic ganglion cells, Age, 8 (1985) 19-20. 31 Varon, S., Manthorpe, M. and Adler, R., Cholinergic neuronotrophic factors: I. Survival, neurite growth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia, Brain Research, 173 (1979) 29-45. 32 Varon, S., Skaper, S.D. and Manthorpe, M., Trophic activities for dorsal root and sympathetic ganglionic neurons in media conditioned by Schwann and other peripheral cells, Dev. Brain Res., 1 (1981) 73-87. 33 Wood, P.M., Separation of functional Schwann cells and neurons from normal peripheral nerve tissue, Brain Research, 115 (1976) 361-375.
100
Elsevier BRE 11145
Effects of Nerve Growth Factor and Heart Cell Conditioned Medium on Neurite Regeneration of Aged Sympathetic Neurons in Culture YOKO UCHIDA and MASANORI TOMONAGA
Department of Clinical Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2, Sakae-cho, Itabashi-ku, Tokyo-173 (Japan) (Accepted February 5th, 1985)
Key words: nerve growth factor - - heart-cell-conditioned medium - - aging - - sympathetic neuron - neurite regeneration - - cell culture
The effects of nerve growth factor (NGF) and heart-cell-conditioned medium (HCM) on the neurite regeneration of aged sympathetic neurons were investigated in culture. Investigation of HCM was carried out by two different methods: one was the use of whole HCM on collagen substratum, which reflected component(s) effective in solution (HCM-S); the other was the use of polyornithine (PORN)-binding component(s) (P-HCM). Superior cervical ganglion neurons prepared from male mice from 6 to 30 months of age were cultured in MEM-10% FCS on collagen or gelatin-PORN substratum for 3 days. The number of neurons with neurites and the length of neurites were quantified as neurite production and elongation, respectively. Neuronal survival was not affected by addition of NGF, HCM-S or P-HCM. Neurite production of early adult neurons was enhanced by NGF, HCM-S or P-HCM. In contrast, neurite production of aged neurons was enhanced by only HCM-S, but not NGF or P-HCM. HCM-S did not promote neurite elongation in neurons at any age. Neurite elongation of early adult neurons was enhanced by NGF or P-HCM. Neurite elongation of aged neurons was enhanced by P-HCM. However, responsiveness of NGF for neurite elongation varied according to substrata. No age-related difference was found in neurite production and elongation in the absence of NGF, HCM-S or P-HCM. These results indicate that responsiveness of aged sympathetic neurons is various in different growth factors.
INTRODUCTION
ability of neurons to respond to growth factors. In a previous study 30, we showed that responsive-
Since the n u m b e r of axons and synapses decrease in aged animals, the regenerative process may be damaged in the aged nervous system. In fact, several studies indicate that axonal sprouting and synaptic formation in aged nervous system decline after partial denervation in viv08,13,23.2s. As an explanation for
ness to N G F was diminished in sympathetic ganglion neurons from aged mice in explant culture. In ganglion explants, however, many n o n - n e u r o n a l cells as
the diminished plasticity in aged animals, the following speculation has been presented28: the aged neurons may have less ability to synthesize or assemble materials necessary for growth; growth-inducing substances may not be readily available; or the target cells may be less able to accept new innervation. In vivo studies have not given a satisfactory explanation of the mechanism of diminished plasticity in aged neurons. Tissue or cell culture technique is useful for understanding these mechanisms4, ~8. However, the reason for this reduction in plasticity is not k n o w n yet. It is, therefore, of interest to determine whether this diminished plasticity is due to a reduction in the
well as neurons exist and release several substances into medium. Therefore, the regenerative response of ganglion explants is possibly affected by not only N G F , but also other factors which are released from explants themselves2, 21. Besides chemically defined substances, such as N G F , fibronectin27 and laminin27, several materials which promote n e u r o n a l survival or neurite outgrowth of embryonic sympathetic neurons have been found in various tissue extracts and conditioned media1,11,12A4A7,32. In some cases, the neurite outgrowth-promoting activity has been shown to bind directly to the polycation substratum and operate only in its substratum-associated condition1,11. In the present study, we examined whether dissociated aged sympathetic n e u r o n s respond to N G F
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B .V. (Biomedical Division)
101 and heart-cell-conditioned medium (HCM) in culture. With regard to the latter, we tested the two different components of HCM: one is effective in solution32, the other is effective in polyornithine (PORN)-binding conditionl,~l. MATERIALS AND METHODS Media and reagents Media and reagents used were: CMF, Ca 2+, Mg 2+free Hank's balanced salt solution; medium I, 90% Eagle's minimal essential medium (MEM), 10% fetal calf serum (FCS, Gibco), 6 mg/ml glucose, 5 pg/ml gentamycin, 10-5 M cytosine arabinoside (Ara-C) and 10-5 M fluorodeoxyuridine (FdU)33; medium II: this was identical to medium I except that Ara-C and FdU were deleted33; NGF, 2.5S nerve growth factor (Collaborative Research); NGF antiserum, rabbit antiserum against 2.5S NGF (Collaborative Research); Heart-cell-conditioned medium (HCM), heart cells were dissociated from 9 days chick embryos, and grown to confluent in M E M - 1 0 % FCS. HCM was obtained by exposing the culture to fresh MEM-10% FCS for 48 h 31.
ture plates precoated with collagen or gelatin-PORN and fed in medium I for 24 h, and then in medium II for a further 48 h (37 °C, 5% CO2-air). To determine the effect of NGF, 10 or 100 ng/ml of NGF was added to medium I and medium II. To test the effect of HCM component(s) which is effective in solution, SCG neurons were fed on collagen substratum in medium I and II, containing HCM (0.5 ml/ml) and NGF antiserum (2.5/A/ml). To determine the effect of PORN-binding component(s) of HCM, gelatinPORN precoated wells were incubated with 1 ml/well of either HCM or M E M - 1 0 % FCS (control) for 24 h at 37 °C in 5% CO2-airk After the medium was removed, wells were washed with fresh M E M - 1 0 % FCS and seeded with SCG cells. The culture was fed in medium I and II. After 3 days in culture, cells were fixed with 2% glutaraldehyde in MEM. All single neurons and the single neurons with neurites at least twice the cell diameter were counted. The neurite length was determined by measuring the distance between the end of the longest neurite and the cell body. Measurement was done for 10 neurons in each well. RESULTS
Substrata Collagen prepared from rat tail tendon9 was spread on culture plates (containing 24 mm diameter wells), and gelled by exposure to NH 3 vaper for 5 min. The culture plates were then washed extensively with MEM. Gelatin-polyornithine (PORN)-coated plates were prepared according to the procedure previously reported 22 except that PORN concentration was reduced to 0.1 mg/ml.
The living cell populations at the time of isolation were 72 _+ 5%, 73 _+ 5% and 70 _+ 5% from mice 6, 18 and 24-30 months of age, respectively. No difference was detected in living cell population among the 3 groups.
100
Neuronal cell culture Superior cervical ganglia (SCG) were dissected from male C57BL/6 mice 6, 18 and 24-30 months of age. Decapsulated ganglia were treated with CMF containing 0.25% trypsin-collagenase at 37 °C for 15 rain. At the end of the incubation, M E M - 1 0 % FCS was added to stop digestion. Then ganglia were re-digested with 0.25% collagenase in CMF at 37 °C for 2 h. Dissociation was carried out in M E M - 1 0 % FCS by gentle aspiration using a heat-polished, glass Pasteur pipette. SCG neurons (1600 cells/well) were seeded on cul-
A
[] 0 ng/m[ [ ] 'tO nglml • 100 ng/ml
B
g
"650 i 6
18
24-30 6 Months of age
18
24-30
Fig. 1. Effect of NGF on survival of SCG neurons from mice 61 18 and 24-30 months of age at 3 days in culture on the collagen (A) or gelatin-PORN (B). Each value represents the mean + S.E.M. of 5 determinations.
102 Neuronal response to N G F As Fig. 1 shows, neuronal survival at 3 days in culture on the collagen was slightly higher than that on the gelatin-PORN substratum, but not significantly different (P > 0.1). Neuronal survival was not affected by either the age of the neuron or addition of N G F (Fig. 1). The neurite production, expressed as a percentage of neurons with neurites, on gelatin-PORN was significantly higher (about 3-fold) than that on the collagen substratum in all age groups with or without of N G F (Fig. 2). As shown in Fig. 2, no difference was found in neurite production among 3 groups of neurons cultured without N G F on either substratum. N G F enhanced the neurite production of 6-month neurons, but not of neurons 18 months or more on collagen or gelatin-PORN substratum (Fig. 2). As Fig. 3 shows, the neurite elongation, expressed as neurite length, of neurons of 18-months or more, was almost the same as that of 6-month neurons in the absence of N G F on either substratum. Whereas neurite elongation of 6-month neurons was enhanced by addition of N G F on either substratum, it was somewhat different in aged neurons. Neurite elongation of neurons of 18-months or more was enhanced by N G F on the collagen (Fig. 3A), but not on the gelatin-PORN (Fig. 3B). Neuronal response to H C M component(s) in solution (HCM-S) As shown in Fig. 4A, HCM-S did not increase the
[] []
3O
0 ng/ml 10 ng/ml
==
.,ooze,m,
t2O
.o
B
i ll
A
[] [] •
v
B
0 ng/mt 10 nglrnl 100 ng/ml
5OO
250 o) Z
0 18
6
24-30
6
18
24-30
Months of ccje
Fig. 3. Effect of N G F on neurite length of SCG neurons from mice 6, 18 and 2 4 - 3 0 m o n t h s of age at 3 days in culture on the collagen (A) or gelatin-PORN (B). Each value represents the mean _+ S.E.M. of 5 determinations. Significant differences from control (NGF 0 ng/ml) at P < 0.05 (*) and P < 0.005
(***).
number of surviving neurons in any age group. The neurite production in all age groups was enhanced in the presence of HCM-S (Fig. 4B). This component did not enhance the neurite elongation of early adult or aged neurons (Fig. 4C).
[] []
6O
"6
Control HCM
30
0
c c
5~
2°f ~o 0
400
~o -~
"6
200
0 6
18
24-30
6
18
2/.-30
Months of age
Fig. 2. Effect of N G F on percentage of SCG neurons with neurite. Neurons from mice 6, 18 and 24-30 m o n t h s of age were cultured on the collagen (A) or gelatin-PORN (B) for 3 days. Each value represents the m e a n + S.E.M. of 5 determinations. Significant differences from control ( N G F 0 ng/ml) at P < 0.05 (*) and P < 0.025 (**) estimated using Student's t-test.
6
18
24-30
Months of acje
Fig. 4. Effects of H C M on survival (A), percentage of neurons with neurite (B) and the length of neurite (C) of SCG neurons of various ages at 3 days in culture on the collagen. Each value represents the m e a n _+ S.E.M. of 5 determinations. Significant differences from control at P < 0.05 (*) and P < 0.005 (***).
103
Neuronal response to PORN-binding component(s) of HCM (P-HCM) As Fig. 5A shows, P - H C M did not increase the neuronal survival in any age group with or without NGF. The neurite production was enhanced by PH C M in 6-month neurons, but not in neurons of 18months or more (Fig. 5B). The combination of PH C M and N G F increased the neurite production of both 6- and 18-month neurons (Fig. 5B). Nevertheless, aged neurons (24-30 months of age) did not increase their neurite production in the presence of both P - H C M and N G F (Fig. 5B). On the other hand, the neurite elongation was enhanced drastically by the addition of P - H C M in all age groups (Figs. 5C and 6). The combination of P - H C M and N G F also enhanced the neurite elongation of all age groups (Fig. 5C). DISCUSSION The results indicate that: (1) neuronal survival is not enhanced by either N G F or H C M in any age groups; (2) the responsiveness to N G F for both neurite production and elongation on P O R N substratum declines in aged neurons; (3) H C M component(s) in solution enhances the neurite production but not the elongation in any age groups; (4) responsiveness of neurons to PORN-binding component(s) of H C M for
A
[] [] []
6C
Controt ~-HCM F-HCM ÷ NGF
J
20
13 -
30
c .c
~c
2o
o
2
-
I
10
c
6
18
M o n t h s of a g e
24-30
6
18
1
24-30
M o n t h s of a g e
Fig. 5. Effects of PORN-binding fraction of HCM on survival (A), percentage of neurons with neurite (B), and neurite length (C) of SCG neurons from various age mice at 3 days in culture. Each value represents the mean + S.E.M. of 5 determinations. Significant differences from control at P < 0.05 (*), P < 0.025 (**) and P < 0.005 (***).
neurite production but not neurite elongation, decreases with age. The second result is somewhat different from our previous observation30 that neurite elongation of SCG neurons from mice 24 months of age was enhanced by a high concentration of N G F in explant culture. This discrepancy may be because of the difference in culture method (explant culture vs dissociation culture). In explant culture, high concentration of N G F may promote the release of some kind of neurite-promoting substances into media from numerous neuronal and non-neuronal cells. In fact, ganglion itself produces substances that act as neurite promoting factors by binding to the substrate surrounding the ganglion2, 21. In dissociation culture, because of low cell density (1600 cells/24 m m well), little substances may be released. Other possible causes are that the aged neurons might be damaged more severely by enzymatic dissociation, or be less capable of recovery from injury. Trypsin is reported to inactivate N G F receptor of the m e m b r a n e fraction of SCG 5. However, it should be noted that: (1) the survival of aged neurons was almost the same as that of early adult neurons; (2) even aged neurons have the ability to respond to N G F for neurite elongation on collagen substratum. Our observation also indicates the following influences of substratum on the neurite regeneration: (1) gelatin-PORN supports more extensive neurite regeneration than collagen does with or without N G F regardless of neuronal age; and (2) neurite elongation of aged neurons is enhanced by N G F on the collagen but not on the gelatin-PORN. The former conclusion is consistent with an earlier study by Hawrot 22. The latter difference, based on substratum, cannot be explained at present. It might reflect that the promotion of neurite elongation by N G F was affected by cell surface materials involved in cellP O R N interaction 10, and the synthesis of these materials was suppressed in aged neurons. N G F is a protein essential for survival, development and differentiation of embryonic peripheral sympathetic neurons both in vivo and in vitro. Moreover, N G F plays a role in maintenance of adrenergic function in adult sympathetic neurons 3,7,19,20,26. However, the issue of whether adult sympathetic neurons depend on N G F for survival in vivo is not consistent 19,20,26. In culture of sympathetic neurons, N G F
104
Fig. 6. Phase-contrast micrographs of SCG neurons from mice 6 (a, c, e) and 30 (b, d, f) months of age, cultured for 3 days in the absence (a, b, e, f) and presence (c, d) of NGF. The gelatin-PORN substratum was pretreated before cell seeding with either fresh MEM-FCS medium (a, b, c, d) or heart-cell conditioned medium (e, f).
105 becomes less and less required for cell survival at later embryonic stages 29. O u r observations agree with this conclusion that N G F - d e p e n d e n c y for survival decreases in adult neurons. N G F is r e p o r t e d to exert a stimulatory effect on the regrowth of adrenergic fibers in the young adult (less than 3 months of age) after 6 - O H D A lesioning in vivo6. H o w e v e r , little is known about the action of N G F on neurite regeneration in adult or aged neurons. W e provided the first evidence that: (1) N G F p r o m o t e s both neurite production and elongation in adult neurons; but (2) the action of N G F for neurite production decreases with aging. It remains to be det e r m i n e d whether decreased responsiveness to N G F in aged neurons is due to the alteration in r e c e p t o r site or intercellular mechanisms which lead to neurite regeneration. Recently, several kinds of n e u r o n o t r o p h i c or neurite-promoting factors for peripheral ganglionic neurons are found in various tissue extracts and cell-conditioned media. Some of them are b l o c k e d by antiserum against NGF15, 24, but others are effective, even in the presence of anti-NGFl,ll,12,14,17,32. Since none of the factors are chemically defined, some questions arise: (1) is H C M - S actually N G F itself?; and (2) are H C M - S and P - H C M used in this study identical to the substances previously reportedkll,32? It is unlikely that H C M - S is itself N G F , because antiserum against mouse 2.5S N G F was a d d e d to block the activity of N G F in H C M . It should be stressed that species difference was not critical element, since antiserum to mouse 2.5S N G F effectively blocks the N G F - l i k e activity in the chick irides 16. Moreover, the effect of H C M - S on adult neurons is
REFERENCES 1 Adler, R., Manthorpe, M., Skaper, S.D. and Varon, S., Polyornithine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons, Brain Research, 206 (1981) 129-144. 2 Adler, R. and Varon, S., Neuritic guidance by polyornithine-attached materials of ganglionic origin, Dev. Biol., 81 (1981) 1-11. 3 Angeletti, P.V., Levi-Montalcini, R. and Caramia, F., Analysis of the effect of the antiserum to nerve growth factor in adult mice, Brain Research, 27 (1971) 343-355. 4 Argiro, V. and Johnson, M.I., Patterns and kinetics of neurite extension from sympathetic neurons in culture are age dependent, J. Neurosci., 2 (1982) 503-512.
not different from that of N G F , namely, N G F but not H C M - S p r o m o t e s the neurite elongation. The factors in H C M - S and P - H C M may correspond to the factors r e p o r t e d by V a r o n et al. 32 and A d l e r et al.1., respectively, because H C M - S and PH C M were p r e p a r e d from the same source by the same methods as the)' used. On the other hand, they used embryonic ganglion neurons, whereas we used adult neurons to examine the effect of trophic factors. The possibility could not be ruled out that the factors in H C M which we used were different from those previously r e p o r t e d 1,11,32, because the requirement of cultured sympathetic neurons for trophic factors changes during d e v e l o p m e n t 17. The isolation and characterization of H C M - S and P - H C M should provide the information on the similarity or dissimilarity among those factors. In conclusion, adult mouse sympathetic neurons kept in culture respond to N G F or H C M factors for neurite regeneration. H o w e v e r , in the aged neurons, the responsiveness to these factors varies between different growth factors, or different aspects of regeneration, such as neurite production or elongation. In other words, some, mechanisms for neurite regeneration may be disordered, but others may normally act in aged neurons. ACKNOWLEDGEMENTS This work was s u p p o r t e d in part by the Institute of Physical and Chemical Research as a part of the Project on Discovery of factors Regulating Aging. W e wish to thank Dr. F. Mizobe for his helpful discussions.
5 Banerjee, S.P., Cautrecasas, P. and Snyder, S.H., Nerve growth factor receptor binding, J. Biol. Chem., 250 (1975) 1427-1433. 6 Bjerre, B., Bjorklund, A. and MoNey, W., A stimulatory effect by nerve growth factor on the regrowth of adrenergic nerve fibers in the mouse peripheral tissues after chemical sympathectomy with 6-hydroxydopamine, Z. Zellforsch., 146 (1973) 15-43. 7 Bjerre, B., Wiklung, L. and Edwards, D.C., A study of the de- and regenerative changes in the sympathetic nervous system of the adult mouse after treatment with antiserum to nerve growth factor, Brain Research, 92 (1975) 257-278. 8 Black, M.M. and Lasek, R.J., Slowing of the rate of axonal regeneration during growth and maturation, Exp. Neurol., 63 (1979) 108-119.
106 9 Bornstein, M.B., Reconstituted rat-tail collagen used as substrate for tissue cultures on coverslips in maximow slides and roller tubes, Lab. Invest., 7 (1958) 134-137. 10 Chernoff, E.A.G., Maresh, G.A. and Culp, L.A., Isolation and characterization of a large, neurite-associated glycoconjugate from neuroblastoma cells, J. Cell Biol., 96 (1983) 661-668. 11 Collins, F., Induction of neurite outgrowth by a conditioned-medium factor bound to culture substratum, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 5210-5213. 12 Collins, F., Neurite outgrowth induced by the substrate associated material from nonneuronal cells, Dev. Biol., 79 (1980) 247-252. 13 Cotman, C.W. and Scheff, S.W., Compensatory synapse growth in aged animals after neuronal death, Mech. Aging Develop., 9 (1979) 103-117. 14 Coughlin, M.D., Bloom, E.M. and Black, I.B., Characterization of a neuronal growth factor from mouse heart-cellconditioned medium, Dev. Biol., 82 (1981) 56-68. 15 Crutcher, K.A. and Collins, F., In vitro evidence for two distinct hippocampal growth factors: basis of neuronal plasticity?, Science, 217 (1982) 67-68. 16 Ebendal, T., Hedlund, K.-O. and Norrgren, G., Nerve growth factors in chick tissues, J. Neurosci. Res., 8 (1982) 153-164. 17 Edgar, D., Barde, Y.-A. and Thoenen, H., Subpopulations of cultured chick sympathetic neurons differ in their requirements for suvival factors, Nature (London), 289 (1981) 294-295. 18 Fukuda, J. and Yamaguchi, K., Nerve cells of senescent mouse grown in tissue culture, Neurosci. Lett., 28 (1982) 331-335. 19 Goedert, M., Otten, U. and Thoenen, H., Biochemical effect of antibodies against nerve growth factor on developing and differentiated sympathetic ganglia, Brain Research, 148 (1978) 264-268. 20 Gorin, P.D. and Johnson, E.M., Effects of long-term nerve growth factor deprivation on the nervous system of the adult rat: an experimental autoimmune approach, Brain Research, 198 (1980) 27-42. 21 Gundersen, R.W. and Barrett, J.N., Neurite growth conesubstratum adherance increases in vitro, Dev. Brain Res., 12 (1984) 21-26.
22 Hawrot, E., Cultured sympathetic neurons: effects of cellderived and synthetic substrata o n survival and development, Dev. Biol., 74 (1980) 136-151. 23 Hulsebosch, C.E. and Coggeshall, R.E., Age related sprouting of dorsal root axons after sensory denervation, Brain Research, 288 (1983) 77-83. 24 Lindsay, R.M., Adult rat brain astrocytes support survival of both NGF-dependent and NGF-insensitive neurones, Nature (London), 282 (1979) 80-82. 25 McWilliams, J.R. and Lynch, G., Synaptic density and axonal sprouting in rat hippocampus: stability in adulthood and decline in late adulthood, Brain Research, 294 (1984) 152-156. 26 Otten, U., Goedert, M., Schwab, M. and Thibault, J., Immunization of adult rats against 2.5 S NGF: effects on the peripheral sympathetic nervous system, Brain Research, 176 (1979) 79-90. 27 Rogers, S.L., Letourneau, P.C., Palm, S.L., McGarthy, J. and Furcht, L.T., Neurite extension by peripheral and central nervous system neurons in response to substratumbound fibronectin and laminin, Dev. Biol., 98 (1983) 212-220. 28 Scheff, S.W., Bernardo, L.S. and Cotman, C.W., Decrease in adrenergic axon sprouting in the senescent rat, Science, 202 (1978) 775-778. 29 Selak, I., Skaper, S.D. and Varon, S., Ionic behavior and neuronal survival in developing ganglia: III. Studies with embryonic chick sympathetic neurons, J. Cell Physiol., 114 (1983) 229-234. 30 Uchida, Y. and Tomonaga, M., Nerve growth factor accelates regeneration of cultured adult sympathetic ganglion cells, Age, 8 (1985) 19-20. 31 Varon, S., Manthorpe, M. and Adler, R., Cholinergic neuronotrophic factors: I. Survival, neurite growth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia, Brain Research, 173 (1979) 29-45. 32 Varon, S., Skaper, S.D. and Manthorpe, M., Trophic activities for dorsal root and sympathetic ganglionic neurons in media conditioned by Schwann and other peripheral cells, Dev. Brain Res., 1 (1981) 73-87. 33 Wood, P.M., Separation of functional Schwann cells and neurons from normal peripheral nerve tissue, Brain Research, 115 (1976) 361-375.