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Gangliosides of the mouse spinal cord: A comparison in in vivo and in vitro tissues
0736--5748/89$03.00+0.00 Pergamon Pressplc. ISDN
Int. J. Devl. Neuroscience, Vol.7, No. I, pp. 93-101, 1989.
Printed in Great Britain.
GANGLIOSIDES OF THE MOUSE SPINAL CORD: A COMPARISON IN IN V I V O A N D IN V I T R O TISSUES R. E. BAKER,* B. GUC:ROLDtand H. DREYFUSt *Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands and Centre de Neurochimie du CNRS and Unit~ 44 INSERM, 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France (Received l February 1988;in revised form 9 August 1988;accepted 31 August 1988)
Abstract--43anglioside profiles in spinal cord from 13-day mouse fetuses, 21-day postnatal and adult mice were compared with those harvested from organotypic cross-sections of fetal mouse spinal cord grown for 28 days in vitro in a serum-free medium. All the major species of gangliosides reported for brain were present both in the in vivo tissue and cultured spinal cord, though not necessarily at each developmental stage examined. Fresh tissues showed increases and decreases in various gangliosides as have been reported for higher brain centers at similar stages of development in mammals and birds. However, qualitative and quantitative differences exist between fresh spinal cord and cultured cord explants as well as between galactose-grownand galactose-free cultures. Spinal cord explants grown in the presence of galactose showed measurable amounts of GM2 and GM3 which were not detected in the control-defined medium-growncultures. The differences between the two culture groups may be related to interneuronal connectivitypatterns. Key words: Ganglioside, Development, Spinal cord, Mouse, Tissue culture.
The amount, distribution and composition of gangliosides in the vertebrate CNS have been reported in many species during development. 19 A general developmental feature in gangliosides reported for cerebral cortex and cerebellum is for there to be an increase in the complexity of the oligosaccharide chains extending into the extracellular space. Similar changes have also been observed in cortical and retinal tissues grown in v i t r o . 8"17 Since these changes take place during periods of neuritogenesis and synaptogenesis the more complex gangliosides might play a role in initiating or maintaining one or both of these processes. It has been suggested that the strategic location of the gangliosides on the outer surface of the neural membranes, coupled with the great variability possible in the configuration of their oligosaccharide chains, makes these molecules excellent candidates for selective intercellular recognition and/or adhesion molecules. 19 In contrast with the brain, nothing is known about ganglioside levels in the spinal cord during development. This is an important omission since there is some evidence that gangliosides are involved in the formation of selective connections between D R G and SC maintained in v i t r o , i U e n o et al. 33 found lower ganglioside levels in adult cat, rabbit and human spinal cord than in cerebral cortex, but all of the major gangliosides reported for the cortex were also present in the cord. The profiles, however, were quite different between brain and cord: there was significantly less G D l a , but more GM3 and GD3, in the cord than in cortical tissue. Dawson and Stefansson 7 also observed a gradient of the G D l a ganglioside from low to high in cervical and sacral adult human cord. Organotypic explants of fetal mouse S C - D R G have successfully been used as an in v i t r o model for investigating the role function and environment play in the formation of selective sensory afferent connections. 2'5"6"14'2~Recent developments in culturing media have resulted in the formulation of a CDM, which is a serum-free growth medium capable of maintaining healthy appearing S C - D R G explants for weeks or months. 2 In contrast to serum-grown explants, however, the sensory afferents show no preference for any given region of the spinal cord when grown in the absence of serum. 2 Subsequent studies have shown that the addition of the simple hexose sugar galactose or mixtures of commercially available gangliosides restore the selective preference of the D R G afferents to dorsal regions of the cord. 1.3 It was suggested that galactose brought about the renewed selective preferences by the sensory afferents through its incorporation into the oligosaccharide chains of gangliosides. Abbreviations: CNS, central nervous system; CDM, chemicallydefined medium; DMEM, Dulbecco'smodified Eagle's minimum essential medium; DRG, dorsal root ganglia;NeuAc, N-acetylneuramicacid; SC, spinal cord; SC-DRG, spinal
cord--dorsal root ganglia.
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Nothing is known about the ganglioside content of S C - D R G explants, hence it has not been possible to determine how the quantity or quality of gangliosides in this model might correlate with those of fresh tissue or whether differences exist between explants evincing selectivity and those showing no selective projection patterns. The present study was carried out in order to determine ganglioside profiles in fresh mouse spinal cord and organotypic S C - D R G explants chronically grown in CDM and in a galactose-containing medium known to support the formation of selective sensory afferent projection patterns i n v i t r o . EXPERIMENTAL PROCEDURES Thirty-three 13-day Swiss Random mouse fetuses were removed from the uterus and the spinal cord (stripped of D R G ) rapidly excised. The cords were divided into rostral and caudal halves (at the thoracic midpoint), washed in several changes of 4°C physiological saline, rapidly frozen and freeze-dried. Postnatal (21 day, n = 10) and adult mice (4-5 months old, n = 5) were decapitated and the spinal cord (minus D R G ) rapidly dissected from the vertebral canal. The cords were divided into rostral and caudal portions as above. The divided tissues were then washed in several changes of cold physiological saline, rapidly frozen and freeze-dried. S C - D R G cultures (130 controls and 141 galactose-grown explants) were prepared as described previously (Fig. 1). 2 Briefly, the spinal cord with attached D R G s were removed intact from 13day mouse fetuses and cut into cross-sections of c a 0.5-1.0 mm thick throughout the length of the excised cord. These were then plated onto collagen-coated coverslips of Petri dishes (35 mm with air vents) and grown for 27-28 days in a CDM consisting of 3 parts D M E M , 1 part Ham's F-12, with 200 I~g/ml transferrin, 200 I~M putrescine, 40 nM progesterone, 60 nM selenium, l0 I~g/ml insuline and c a 0.1% albumin (total protein). Experimental cultures received, in addition, 10 l~g/ml galactose-l-phosphate. All culture dishes were placed on grids over sterile distilled water and housed in stainless steel boxes in culturing ovens at 35°C, continuously gassed with 95% air and 5% CO2. Explants were refreshed once a week thereafter, at which time 100 lxg/ml vitamin C and 25 Ixg/ml glutamine were added to the refreshing medium. At harvesting, the explants were washed with two changes of cold physiological saline, rapidly frozen and freeze-dried. The different samples were pooled per group, weighed and then dispersed by sonication in 0.5 ml distilled water. Aliquots were taken for protein determination. TM The lipids of the remaining homogenates were extracted as described previously. "~ Ganglioside NeuAc was determined by the resorcinol method of Svennerhoim, 3° using butanol-butyl acetate (15:85, v/v) to extract the chromophore. 23 The extraction of the blue color was read at 580 nm in a Vernon spectrophotometer, using free sialic acid as the standard. One-dimensional ascending chromotography, sufficient to separate and identify small amounts of ganglioside NeuAc (3-4 I~g), was performed on high performance thin layer chromatographic silica plates (Merck, 5641) in three successive solvents (Fig. 2). "~The gangliosides were identified by co-chromatography with reference substances (pure gangliosides or a mixture of gangliosides from bovine brain to which GM2 and GM3 were added). The relative distribution was established by densitometric scanning at 580 nm using Camag densitometer. From the total ganglioside content and the relative distribution the total amount of each ganglioside was calculated. The gangliosides were named according to Svennerhoim's nomenclature. 3~ The Friedman test was used to determine statistical differences between the groups using P < 0 . 0 5 as the level of confidence. RESULTS The total amount of protein and ganglioside NeuAc was observed to increase with age in the fresh tissue (Table 1). Rostral spinal cord showed slightly higher levels of NeuAc per mg protein than did the caudal cord in the fetal and adult groups. The total amount of protein and ganglioside NeuAc was considerably smaller in the two culture groups, whereas the level of NeuAc per mg protein was 1.5 times that of fresh tissues (Table 2). All of the common gangliosides were present in rostral and caudal cord but their amounts and distributions differed between the three groups (Figs 3 and 4). Fetal spinal cord contained GM3
Gangliosides of m o u s e spinal cord
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Fig. 1. Scanning electron micrograph of a SC-DRG explant grown for 25 days in vitro in a galactosecontaining medium. DRG, dorsal root ganglion; SC, spinal cord; d, dorsal side of the cord explant; v, ventral side; c, central canal. Arrow points to the connecting bundle of sensory afferents.
ii,i,,~ ~
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2--i ~
~:::~ii::i::i~i~"::~,
--4-5m
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Fig. 2. HPTLC of gangliosides isolated from 13-day fetal (F13) and adult (Ad) mouse spinal cord. R = reference gangliosides from Cronassial (Fidia Research Laboratories, Abano Terme, Italy) to which pure GM2 and GM3 were added. Gangliosides were named according to Svennerholm 31 and Dreyfus et al. "} for GT1L. 1=GM3; 2 = G M 2 ; 3 = G M 1 ; 4 = G D 3 ; 5 = G D l a ; 6 = G D l b ; 7=GT1L; 8 = G T l b ; 9 = GQ.
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Gangliosides of mouse spinal cord Table i. Protein and ganglioside amounts in fetal, postnatal and adult mouse spinal cord Proteins (mg)
Ganglioside NeuAc (~,g)
Ganglioside NeuAe/prot. (Izg/mg)
22.1 15.5
41.8 26.9
1.9 1.7
19.6 19. I
49.2 47.8
2.5 2.5
35.4 49.6
84.5 110.7
2.4 2.2
Fetal group rostral caudal Postnatal group rostral caudal Adult group rostral caudal
Table 2. Protein and ganglioside amounts in control and galactose-grown SC-DRG cultures Proteins (mg)
Ganglioside NeuAc (~,g)
Ganglioside NeuAc,/prot. (Izg/mg)
1.4 1.6
4.5 5.6
3.2 3.4
CDM group Galactose group
while b o t h t h e 21-day p o s t n a t a l a n d a d u l t tissues s h o w e d n o d e t e c t a b l e a m o u n t s o f this g a n g l i o s i d e (Fig. 3). In c o n t r a s t , t h e r e was no d e t e c t a b l e a m o u n t o f G M 1 a n d G T 1 L (a p r o b a b l e i s o m e r o f G T l b ) ' " in the fetal c o r d . G M 1 was p r e s e n t in b o t h the 21-day a n d a d u l t m o u s e spinal c o r d , s h o w i n g a s h a r p i n c r e a s e b e t w e e n t h e two g r o u p s , with c a u d a l c o r d c o n t a i n i n g g e n e r a l l y h i g h e r levels t h a n r o s t r a l c o r d . G T I L was o n l y p r e s e n t in p o s t n a t a l a n d a d u l t c o r d , with o n l y the 1200 =
-
Rostral
o. . . . o Caudal
./~..~/.,/.
/
GM1
o
GM1
1000
0_ o~
800
E ID
.~- 600 t.-
o ~ 40O E •
o GM3 ..............
200
i
13 day Fetus n=33
[11;
. . . . . o GD3 ~.~.~ GT1L GT1L IGD3 GQ .......... GQ
i
21 d a y Neonate n=10
i
Adult n=5
Fig. 3. Developmental profiles of gangliosides in the mouse spinal cord.
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ROSTRAL []
[]
800
[]
CAUDAL
GDla GDlb GTlb
c_
n
600
E ~3
~
4oo
c
E
200
o.
13 day Fetus
21 day Neonate
Adult
13 day Fetus
21 day Neonate
Adult
Fig. 4. G D I a , G D I b and G T I b content in mouse spinal cord. Values in caudal spinal cord of fetuses were significantly different from those of the postnatal animals ( P < 0 . 0 1 ) and adults ( P < 0 . 0 5 ) .
caudal portions of the cord showing increased amounts in the adult tissues (Fig. 3). The amount of GQ remained stable from the fetus through to the adult. There was a 3-fold decrease in the amount of GD3 from fetal to postnatal cord, which continued to decrease into the adult. The levels of GD3 were also consistently greater in the caudal spinal cord than in the rostral cord. GDla, G D l b and GTIb increased from fetal to postnatal stages and then gradually declined in the adult (Fig. 4). These changes occurred in both rostral and caudal portions of the cord, with the caudal regions generally showing higher amounts than the rostral regions. There was a significant difference among the three age groups in the total amount of these three gangliosides for the total cord ( P < 0.01 postnatal vs fetal; P < 0.05 adult vs fetal values, two-tailed). This difference could also be seen in the caudal portion of the cord while the amounts of the three gangliosides in the rostrai cord fell just below the level of significance. All but one of the major gangliosides (GM2) found in fresh tissue were found in both culture groups. However, the amount and distribution of several species differed between age-matched 21-day postnatal tissue and the two culture groups (Table 3). Moreover, the CDM controls and the experimental galactose-grown cultures also differed from one another. The total nmol ganglioside NeuAc per mg protein was higher in the galactose group due to the presence of the monosialogangliosides GM3 and GM2. There were no detectable amounts of either of these two Table 3, Ganglioside amounts in S C - D R G explants chronically exposed to a serum-free medium Control group GM3 GM2 GM I GD3 GDIa GDIb GTI b GTIL GQ Total
1).3 0.7 1.0 1.1 I, 1 -0.3 4.3
Galactose group 1.8 O.4 0.4 0.7 1).9 0.7 09 0.3 6.~]
2 l-day postnatal* group
0.9 0.4 (I.75 0.7 0.65 0.25 (1.2 3.85
* Each value represents the mean of rostral and caudal halves of the cord. Results are expressed as nmol ganglioside Neu-Ac/mg protein.
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gangliosides in the postnatal and CDM groups using the present methods of separation and analysis. The level of GQ and GD3 was the same in both culture groups. Three gangliosides, G D l a , G D l b amd GTlb represented the majority of the gangliosides in both groups, 2.5 and 3.2 nmol/mg protein for the galactose and CDM groups, respectively. There was no significant difference, however, between the ganglioside profiles in the two culture groups (Friedman test). DISCUSSION The present study shows that mouse spinal cord contains all of the major gangliosides described for brain tissue and that the same gangliosides are present in organotypic explants of the cord after several weeks in vitro. The ganglioside profile of the mouse spinal cord changes qualitatively and quantitatively during development, these changes being similar to those described for higher brain c e n t e r s . 22'24"25"~ The consistently higher levels of practically all the major gangliosides in caudal vs rostral postnatal and adult cord, in particular GDla, G D l b and GTlb, may reflect regional differences in cell types or numbers. Regional variation in type and amount of gangliosides occurs throughout the adult human brain. '~ These variations may extend to the individual cells, as has been suggested to occur in murine cerebellum. 26'27 It is not, therefore, unreasonable to presume that similar variations occur in the spinal cord and may, in turn, be involved in the formation of selective cell-cell connections. The present study has shown that the development of ganglioside profiles in organotypic explants of fetal mouse SC-DRG was similar in many respects to that observed in vivo, signifying that the normal progression of changes in these compounds can proceed independently in vitro. Similar parallel changes in ganglioside profiles have been reported in the retina ~7J8 and chick cerebrum 9"*' suggesting that such changes are related to specific aspects of differentiation and maturation of nervous tissues both in vivo and in vitro. Although the explants were grown with attached DRG, which were not included in the fresh tissue examined, the types of gangliosides present in DRG are the same as those observed in cord tissues. 13Moreover, since the fresh tissues contained DRG fiber tracts and terminations, the ganglioside differences observed between the cultured and fresh tissues were probably not simply due to the lack of DRG cell bodies in the latter group. The only qualitative differences measured between the two culture groups were the presence of GM3 and GM2 in the galactose-grown explants where they represented 37% of the ganglioside content in these cultures. High levels of these gangliosides are normally associated with glial cells, 8 however, smaller amounts of GM3 and GM2 have been found in pure neuronal cultures, showing that both can be normal constituents of nerve cells as well. 9J2 It is not known whether, or in what respect, the two culture groups may differ in either their neural or their glial content, making it impossible to speculate on the cellular source of either ganglioside based on the present data. However, the appearance of these two compounds appears to be dependent on the presence of galactose in the culturing medium. The fate of the exogenously added galactose is unknown, though our use of galactose-l-phosphate suggests that it may be incorporated locally, at the cell s u r f a c e . 28'32 The restriction of GM3 and GM2 to cultures which exhibit sensory afferent selectivity suggests that these gangliosides may be in some way associated with this phenomenon. GM2 has been suggested as a possible selectivity factor in developing chick retinotectal adhesion, 2~ while Blackburn et al. 4 have shown this ganglioside to be one of the most adhesive of the major gangliosides in the chick neural retina. Although GM2 and GM3 have been shown to occur in immature neuronal tissues in vivo as well as in vitro, their levels decrease with maturation and may even be missing in mature neurons, x This may account for the lack of detectable amounts of these gangliosides in both postnatal and adult cord tissues in the present study. Why GM2 and GM3 are present in vitro but not detected in vivo is unknown. If either or both gangliosides are involved in selectivity, then fresh SC-DRG, which evince selective projection patterns, ought to contain measurable amounts of both. One possible explanation for these differences may be related to the levels of bioelectric activity in the two situations. Hinrichs et al.'5 have shown that increased depolarization of cerebellar neurons in vitro leads to elevated levels of GM1, GM2, GM3, and GD3, but not the major di- and trisialo gangliosides. CDMogrown explants show increasing levels of spontaneous bioelectric discharges with time in vitro, 2 which, in turn, may
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account for the presence of GM2 and GM3 in those cultures grown in the presence of the most appropriate chemical substrates. It was interesting to note that the levels of G D l a , G D l b and G T l b were higher in the CDM controls than in either the galactose-grown group or age-matched fresh tissues. These arc gangliosides presumed to be associated with differentiation and maturation of nervous tissues in vivo and in vitro, particularly with the formation of synapses? ~~ The amounts of these gangliosides in the galactose-grown explants, on the other hand, were similar to the age-matched fresh tissues. These data suggest that differentiation and maturation is occurring in both the m vitro groups, and that those grown in the presence of galactose most closely resemble agematched tissues. Levels of GM1 and GD3 were similar in both groups of cultures. The amount of G M l was less than that found in neonatal (and adult) spinal cord, but this would be expected in an in vitro situation where reduced levels of myelination are occurring. GM1 is a normal constituent of neurons ~2 and it is probable that the amounts measured in the current study represent the G M l present in D R G and spinal nerve cells. The high levels of GD3 in both culture groups may be indicative of increased mitotic activity in the cultures, perhaps associated with glial production. 26.27 The data obtained in the current study has established ganglioside profile patterns for mouse spinal cord at a fetal and postnatal stage of development corresponding to explantation and harvesting of S C - D R G explants in vitro. Since D R G afferents evince a selective preference for dorsal regions of galactose-grown cord explants, regional ganglioside differences may exist in this plane as well as in the rostrocaudai axis. It would now be of interest to examine dorsal and ventral halves of the galactose-grown cord explants and thereby determine whether there are dorsoventral differences in any of the gangliosides present in these cultures which might indicate that these compounds could also be contributing to selective projection patterns as has been suggested for in the visual system. 2~ In addition, if such observations could also be made in SCD R G grown in other serum-free media which do or do not support sensory afferent selectivity, it might be possible to correlate selectivity with the quality or quantity of the gangliosides present in these tissues. A c k n o w l e d g e m e n t s - - T h e authors wish to express their appreciation to Drs M. A. Corner, J. M. Ruijter, M. Mirmiran and H. Romijn for their helpful comments on the manuscript and project. We also thank Mr H. Stoffels for his art work on the two figures and Mr G. van der Meulen for his photographic assistance.
REFERENCES 1. Baker R. E. (1983) Effects of gangliosides on the development of selective afferent connections within fetal mouse spinal cord explants. Neurosci. Lett. 41, 81-84. 2. Baker R. E., Habets A. M. M. C., Brenner E. and Corner M. A. (1982) lnfluence of growth medium, age in vitro and spontaneous bioelectric activity on the distribution of sensory ganglion-evoked activity in spinal cord explants. Devl Brain Res. 5, 329-341. 3. Baker R. E., Corner M. A. and Kleiss M. (1983) Effects of chemical additives on functional innervation patterns in mouse spinal cord-ganglion explants in serum-free medium. Neurosci. Lett. 41,321-324. 4. Blackburn C. C., Swank-Hill P. and Schnaar R. L. 0986) Gangliosides support neural retina cell adhesion. Y. bioL Chem. 261, 2873-2881. 5. Crain S. M. and Peterson E. R. (1967) Onset and development of functional interneuronal connections in explants of rat spinal cord-ganglia during maturation in culture. Brain Res. 6, 750-762. 6. Crain S. M., Bornstein M. B. and Peterson E. R. (1968) Maturation of cultured embryonic CNS tissues during chronic exposure to agents which prevent bioelectric activity. Brain Res. 8, 363-372. 7. Dawson G. and Stefansson K. (1984) Gangliosides of human spinal cord: aberrant composition of cords from patients with amyotrophic lateral sclerosis. J. Neurosci. Res. 12, 213-220. 8. Dreyfus H., Louis J. C.. Harth S. and Mandel P. (1980) Gangliosides in cultured neurons. Neuroscience 5, 1647-1655. 9. Dreyfus H., Harth S., Massarelli R. and Louis J. C. (1981) Mechanisms of differentiation in cultured neurons: involvement of gangliosides. In Gangliosides in Differentiation and Neuromuscular Function (eds Raport M. M. and Gorio A.), pp. 151-170. Raven Press, New York. 10. Dreyfus H., Harth S., Guiliani-Debernardi A., Roos M., Mack G. and Mandel P. (1982) Gangliosides in various brain areas of three inbred strains of mice. Neurochem. Res. 7, 477-488. I I. Dreyfus H., Ferret B., Harth S., Gorio A., Durand M., Freysz L. and Massarelli R. (1984) Metabolism and function of gangliosides in developing neurons. J. Neurosci. Res. 12, 31 i-322. 12. Durand M.. Guerold B., Lombard-Golly D. and Dreyfus H. (1986) Evidence for the effects of gangliosides on the development of neurons in primary cultures. In Gangliosides and Neuroplasticity (eds Tettamanti G., Ledeen R. W., Sandhoff K., Nagai Y. and Toffano G.), pp. 295-307. Liviana Press, Padova, Italy. 13. Graves M., Varon S. and McKhann (3. (1969) The effect of nerve growth factor (N(3F) on the synthesis of gangliosides. J. Neurochem. 16, 1533-1541.
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14. Habets A. M. M. C., Baker R. E., Brenner E. and Corner M. A. (1981) Chemically defined medium enhances bioelectric activity in mouse spinal cord-dorsal root ganglion cultures. Neurosci. Len. 22, 51-56. 15. Hinrichs U., Thomsen S., Van Echten G. and Sandhoff K. (1987) Effect of veratrine on ganglioside biosynthesis in cerebellar cultures. In Gangliosides and Modulation of the Neuronal Functions (ed. Rahmann H.), pp. 319-320. Springer-Verlag, Heidelberg. 16. Kracun i., R6sner H., Cosovic C. and Stravljenic A. (1984) Topographical atlas of the gangliosides of the adult human brain. J. Neurochem. 43, 979-989. 17. Landa C. A. and Moscona A. A. (1985) Changes in ganglioside profile in chick embryo retina: studies on tissue and cell cultures, lnt. J. devl Neurosci. 3, 77-88. 18. Landa C. A., Panzetta P. and Maccioni H. J. F. (1984) Biosynthesis of gangliosides in cultured retina from chick embryos. Devl Brain Res. 14, 83-92. 19. Ledeen R. W. (1985) Gangliosides of the neuron. Trends Neurosci. 8, 169-174. 20. Lowry O. H., Rosebrough N. J., Farr D. A. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. 21. Marchase R. B. (1977) Biochemical investigations of retinotectal adhesive specificity. J. Cell Biol. 75, 237-257. 22. Merat A. and Dickerson J. W. T. (1973) The effect of development on the gangliosides of rat and pig brain. J. Neurochem. 20, 873-880. 23. Miettinen T. and Takki-Luukkainen I. T. (1959) Use of butylacetate in determination of sialic acid. Acta Chem. Scand. 13, 856-858. 24. R6sner H. (1982) Ganglioside changes in the chicken optic lobes as biochemical indicators of brain development and maturation. Brain Res. 236, 49-61. 25. Seybold U. and Rahmann H. (1985) Brain gangliosides in birds with different types of postnatal development (nidifugous and didicolous type). Devl Brain Res. 17, 201-208. 26. Seyfried T. N., Miyazawa N. and Yu R. K. (1983) Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant. J. Neurochem. 41,491-505. 27. Seyfried T. N., Bernard D. J. and Yu R. K. (1984) Cellular distribution of gangliosides in the developing mouse cerebellum: analysis using the staggerer mutant. J. Neurochem. 43, 1152-1162. 28. Shur B. D., Vogler M. and Kosher R. A. (1982) Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis. Expl Cell Res. 137, 229-237. 29. Smalheiser N. R., Peterson E. R. and Crain S. M. (1981) Neurites from mouse retina and dorsal root ganglion explants show specific behavior within co-cultured tectum or spinal cord. Brain Res. 208, 499-505. 30. Svennerholm L. (1957) Quantitative estimation of sialic acids--ll. A colorimetric resorcinol-hydrochloric method. Biochim. Biophys. Acta 24, 604-611. 31. Svennerholm L. (1980) Ganglioside designation. In Structure and Function of Gangliosides (eds Svennerholm L., Mandel P., Dreyfus H. and Urban P. F.), p. 11. Plenum Press, New York. 32. Tolvanen M. and Gahmberg C. (3. (1986) In vitro attachment of mono- and oligosaccharides to surface glycoconjugates of intact cells. J. biol. Chem. 261, 9546-9551. 33. Ueno K., Ando S. and Yu R. K. (1978) Gangliosides of human, cat and rabbit spinal cords and cord myelin. J. Lipid Res. 19, 863-871. 34. Vanier M. T., Holm M., Ohman R. and Svennerholm L. (1971) Developmental profiles of gangliosides in human and rat brain. J. Neurochem. 18, 581-592.
DN 7:I-F
Int. J. Devl. Neuroscience, Vol.7, No. I, pp. 93-101, 1989.
Printed in Great Britain.
GANGLIOSIDES OF THE MOUSE SPINAL CORD: A COMPARISON IN IN V I V O A N D IN V I T R O TISSUES R. E. BAKER,* B. GUC:ROLDtand H. DREYFUSt *Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands and Centre de Neurochimie du CNRS and Unit~ 44 INSERM, 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France (Received l February 1988;in revised form 9 August 1988;accepted 31 August 1988)
Abstract--43anglioside profiles in spinal cord from 13-day mouse fetuses, 21-day postnatal and adult mice were compared with those harvested from organotypic cross-sections of fetal mouse spinal cord grown for 28 days in vitro in a serum-free medium. All the major species of gangliosides reported for brain were present both in the in vivo tissue and cultured spinal cord, though not necessarily at each developmental stage examined. Fresh tissues showed increases and decreases in various gangliosides as have been reported for higher brain centers at similar stages of development in mammals and birds. However, qualitative and quantitative differences exist between fresh spinal cord and cultured cord explants as well as between galactose-grownand galactose-free cultures. Spinal cord explants grown in the presence of galactose showed measurable amounts of GM2 and GM3 which were not detected in the control-defined medium-growncultures. The differences between the two culture groups may be related to interneuronal connectivitypatterns. Key words: Ganglioside, Development, Spinal cord, Mouse, Tissue culture.
The amount, distribution and composition of gangliosides in the vertebrate CNS have been reported in many species during development. 19 A general developmental feature in gangliosides reported for cerebral cortex and cerebellum is for there to be an increase in the complexity of the oligosaccharide chains extending into the extracellular space. Similar changes have also been observed in cortical and retinal tissues grown in v i t r o . 8"17 Since these changes take place during periods of neuritogenesis and synaptogenesis the more complex gangliosides might play a role in initiating or maintaining one or both of these processes. It has been suggested that the strategic location of the gangliosides on the outer surface of the neural membranes, coupled with the great variability possible in the configuration of their oligosaccharide chains, makes these molecules excellent candidates for selective intercellular recognition and/or adhesion molecules. 19 In contrast with the brain, nothing is known about ganglioside levels in the spinal cord during development. This is an important omission since there is some evidence that gangliosides are involved in the formation of selective connections between D R G and SC maintained in v i t r o , i U e n o et al. 33 found lower ganglioside levels in adult cat, rabbit and human spinal cord than in cerebral cortex, but all of the major gangliosides reported for the cortex were also present in the cord. The profiles, however, were quite different between brain and cord: there was significantly less G D l a , but more GM3 and GD3, in the cord than in cortical tissue. Dawson and Stefansson 7 also observed a gradient of the G D l a ganglioside from low to high in cervical and sacral adult human cord. Organotypic explants of fetal mouse S C - D R G have successfully been used as an in v i t r o model for investigating the role function and environment play in the formation of selective sensory afferent connections. 2'5"6"14'2~Recent developments in culturing media have resulted in the formulation of a CDM, which is a serum-free growth medium capable of maintaining healthy appearing S C - D R G explants for weeks or months. 2 In contrast to serum-grown explants, however, the sensory afferents show no preference for any given region of the spinal cord when grown in the absence of serum. 2 Subsequent studies have shown that the addition of the simple hexose sugar galactose or mixtures of commercially available gangliosides restore the selective preference of the D R G afferents to dorsal regions of the cord. 1.3 It was suggested that galactose brought about the renewed selective preferences by the sensory afferents through its incorporation into the oligosaccharide chains of gangliosides. Abbreviations: CNS, central nervous system; CDM, chemicallydefined medium; DMEM, Dulbecco'smodified Eagle's minimum essential medium; DRG, dorsal root ganglia;NeuAc, N-acetylneuramicacid; SC, spinal cord; SC-DRG, spinal
cord--dorsal root ganglia.
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Nothing is known about the ganglioside content of S C - D R G explants, hence it has not been possible to determine how the quantity or quality of gangliosides in this model might correlate with those of fresh tissue or whether differences exist between explants evincing selectivity and those showing no selective projection patterns. The present study was carried out in order to determine ganglioside profiles in fresh mouse spinal cord and organotypic S C - D R G explants chronically grown in CDM and in a galactose-containing medium known to support the formation of selective sensory afferent projection patterns i n v i t r o . EXPERIMENTAL PROCEDURES Thirty-three 13-day Swiss Random mouse fetuses were removed from the uterus and the spinal cord (stripped of D R G ) rapidly excised. The cords were divided into rostral and caudal halves (at the thoracic midpoint), washed in several changes of 4°C physiological saline, rapidly frozen and freeze-dried. Postnatal (21 day, n = 10) and adult mice (4-5 months old, n = 5) were decapitated and the spinal cord (minus D R G ) rapidly dissected from the vertebral canal. The cords were divided into rostral and caudal portions as above. The divided tissues were then washed in several changes of cold physiological saline, rapidly frozen and freeze-dried. S C - D R G cultures (130 controls and 141 galactose-grown explants) were prepared as described previously (Fig. 1). 2 Briefly, the spinal cord with attached D R G s were removed intact from 13day mouse fetuses and cut into cross-sections of c a 0.5-1.0 mm thick throughout the length of the excised cord. These were then plated onto collagen-coated coverslips of Petri dishes (35 mm with air vents) and grown for 27-28 days in a CDM consisting of 3 parts D M E M , 1 part Ham's F-12, with 200 I~g/ml transferrin, 200 I~M putrescine, 40 nM progesterone, 60 nM selenium, l0 I~g/ml insuline and c a 0.1% albumin (total protein). Experimental cultures received, in addition, 10 l~g/ml galactose-l-phosphate. All culture dishes were placed on grids over sterile distilled water and housed in stainless steel boxes in culturing ovens at 35°C, continuously gassed with 95% air and 5% CO2. Explants were refreshed once a week thereafter, at which time 100 lxg/ml vitamin C and 25 Ixg/ml glutamine were added to the refreshing medium. At harvesting, the explants were washed with two changes of cold physiological saline, rapidly frozen and freeze-dried. The different samples were pooled per group, weighed and then dispersed by sonication in 0.5 ml distilled water. Aliquots were taken for protein determination. TM The lipids of the remaining homogenates were extracted as described previously. "~ Ganglioside NeuAc was determined by the resorcinol method of Svennerhoim, 3° using butanol-butyl acetate (15:85, v/v) to extract the chromophore. 23 The extraction of the blue color was read at 580 nm in a Vernon spectrophotometer, using free sialic acid as the standard. One-dimensional ascending chromotography, sufficient to separate and identify small amounts of ganglioside NeuAc (3-4 I~g), was performed on high performance thin layer chromatographic silica plates (Merck, 5641) in three successive solvents (Fig. 2). "~The gangliosides were identified by co-chromatography with reference substances (pure gangliosides or a mixture of gangliosides from bovine brain to which GM2 and GM3 were added). The relative distribution was established by densitometric scanning at 580 nm using Camag densitometer. From the total ganglioside content and the relative distribution the total amount of each ganglioside was calculated. The gangliosides were named according to Svennerhoim's nomenclature. 3~ The Friedman test was used to determine statistical differences between the groups using P < 0 . 0 5 as the level of confidence. RESULTS The total amount of protein and ganglioside NeuAc was observed to increase with age in the fresh tissue (Table 1). Rostral spinal cord showed slightly higher levels of NeuAc per mg protein than did the caudal cord in the fetal and adult groups. The total amount of protein and ganglioside NeuAc was considerably smaller in the two culture groups, whereas the level of NeuAc per mg protein was 1.5 times that of fresh tissues (Table 2). All of the common gangliosides were present in rostral and caudal cord but their amounts and distributions differed between the three groups (Figs 3 and 4). Fetal spinal cord contained GM3
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Fig. 1. Scanning electron micrograph of a SC-DRG explant grown for 25 days in vitro in a galactosecontaining medium. DRG, dorsal root ganglion; SC, spinal cord; d, dorsal side of the cord explant; v, ventral side; c, central canal. Arrow points to the connecting bundle of sensory afferents.
ii,i,,~ ~
i,
2--i ~
~:::~ii::i::i~i~"::~,
--4-5m
7~
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Fig. 2. HPTLC of gangliosides isolated from 13-day fetal (F13) and adult (Ad) mouse spinal cord. R = reference gangliosides from Cronassial (Fidia Research Laboratories, Abano Terme, Italy) to which pure GM2 and GM3 were added. Gangliosides were named according to Svennerholm 31 and Dreyfus et al. "} for GT1L. 1=GM3; 2 = G M 2 ; 3 = G M 1 ; 4 = G D 3 ; 5 = G D l a ; 6 = G D l b ; 7=GT1L; 8 = G T l b ; 9 = GQ.
97
Gangliosides of mouse spinal cord Table i. Protein and ganglioside amounts in fetal, postnatal and adult mouse spinal cord Proteins (mg)
Ganglioside NeuAc (~,g)
Ganglioside NeuAe/prot. (Izg/mg)
22.1 15.5
41.8 26.9
1.9 1.7
19.6 19. I
49.2 47.8
2.5 2.5
35.4 49.6
84.5 110.7
2.4 2.2
Fetal group rostral caudal Postnatal group rostral caudal Adult group rostral caudal
Table 2. Protein and ganglioside amounts in control and galactose-grown SC-DRG cultures Proteins (mg)
Ganglioside NeuAc (~,g)
Ganglioside NeuAc,/prot. (Izg/mg)
1.4 1.6
4.5 5.6
3.2 3.4
CDM group Galactose group
while b o t h t h e 21-day p o s t n a t a l a n d a d u l t tissues s h o w e d n o d e t e c t a b l e a m o u n t s o f this g a n g l i o s i d e (Fig. 3). In c o n t r a s t , t h e r e was no d e t e c t a b l e a m o u n t o f G M 1 a n d G T 1 L (a p r o b a b l e i s o m e r o f G T l b ) ' " in the fetal c o r d . G M 1 was p r e s e n t in b o t h the 21-day a n d a d u l t m o u s e spinal c o r d , s h o w i n g a s h a r p i n c r e a s e b e t w e e n t h e two g r o u p s , with c a u d a l c o r d c o n t a i n i n g g e n e r a l l y h i g h e r levels t h a n r o s t r a l c o r d . G T I L was o n l y p r e s e n t in p o s t n a t a l a n d a d u l t c o r d , with o n l y the 1200 =
-
Rostral
o. . . . o Caudal
./~..~/.,/.
/
GM1
o
GM1
1000
0_ o~
800
E ID
.~- 600 t.-
o ~ 40O E •
o GM3 ..............
200
i
13 day Fetus n=33
[11;
. . . . . o GD3 ~.~.~ GT1L GT1L IGD3 GQ .......... GQ
i
21 d a y Neonate n=10
i
Adult n=5
Fig. 3. Developmental profiles of gangliosides in the mouse spinal cord.
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ROSTRAL []
[]
800
[]
CAUDAL
GDla GDlb GTlb
c_
n
600
E ~3
~
4oo
c
E
200
o.
13 day Fetus
21 day Neonate
Adult
13 day Fetus
21 day Neonate
Adult
Fig. 4. G D I a , G D I b and G T I b content in mouse spinal cord. Values in caudal spinal cord of fetuses were significantly different from those of the postnatal animals ( P < 0 . 0 1 ) and adults ( P < 0 . 0 5 ) .
caudal portions of the cord showing increased amounts in the adult tissues (Fig. 3). The amount of GQ remained stable from the fetus through to the adult. There was a 3-fold decrease in the amount of GD3 from fetal to postnatal cord, which continued to decrease into the adult. The levels of GD3 were also consistently greater in the caudal spinal cord than in the rostral cord. GDla, G D l b and GTIb increased from fetal to postnatal stages and then gradually declined in the adult (Fig. 4). These changes occurred in both rostral and caudal portions of the cord, with the caudal regions generally showing higher amounts than the rostral regions. There was a significant difference among the three age groups in the total amount of these three gangliosides for the total cord ( P < 0.01 postnatal vs fetal; P < 0.05 adult vs fetal values, two-tailed). This difference could also be seen in the caudal portion of the cord while the amounts of the three gangliosides in the rostrai cord fell just below the level of significance. All but one of the major gangliosides (GM2) found in fresh tissue were found in both culture groups. However, the amount and distribution of several species differed between age-matched 21-day postnatal tissue and the two culture groups (Table 3). Moreover, the CDM controls and the experimental galactose-grown cultures also differed from one another. The total nmol ganglioside NeuAc per mg protein was higher in the galactose group due to the presence of the monosialogangliosides GM3 and GM2. There were no detectable amounts of either of these two Table 3, Ganglioside amounts in S C - D R G explants chronically exposed to a serum-free medium Control group GM3 GM2 GM I GD3 GDIa GDIb GTI b GTIL GQ Total
1).3 0.7 1.0 1.1 I, 1 -0.3 4.3
Galactose group 1.8 O.4 0.4 0.7 1).9 0.7 09 0.3 6.~]
2 l-day postnatal* group
0.9 0.4 (I.75 0.7 0.65 0.25 (1.2 3.85
* Each value represents the mean of rostral and caudal halves of the cord. Results are expressed as nmol ganglioside Neu-Ac/mg protein.
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gangliosides in the postnatal and CDM groups using the present methods of separation and analysis. The level of GQ and GD3 was the same in both culture groups. Three gangliosides, G D l a , G D l b amd GTlb represented the majority of the gangliosides in both groups, 2.5 and 3.2 nmol/mg protein for the galactose and CDM groups, respectively. There was no significant difference, however, between the ganglioside profiles in the two culture groups (Friedman test). DISCUSSION The present study shows that mouse spinal cord contains all of the major gangliosides described for brain tissue and that the same gangliosides are present in organotypic explants of the cord after several weeks in vitro. The ganglioside profile of the mouse spinal cord changes qualitatively and quantitatively during development, these changes being similar to those described for higher brain c e n t e r s . 22'24"25"~ The consistently higher levels of practically all the major gangliosides in caudal vs rostral postnatal and adult cord, in particular GDla, G D l b and GTlb, may reflect regional differences in cell types or numbers. Regional variation in type and amount of gangliosides occurs throughout the adult human brain. '~ These variations may extend to the individual cells, as has been suggested to occur in murine cerebellum. 26'27 It is not, therefore, unreasonable to presume that similar variations occur in the spinal cord and may, in turn, be involved in the formation of selective cell-cell connections. The present study has shown that the development of ganglioside profiles in organotypic explants of fetal mouse SC-DRG was similar in many respects to that observed in vivo, signifying that the normal progression of changes in these compounds can proceed independently in vitro. Similar parallel changes in ganglioside profiles have been reported in the retina ~7J8 and chick cerebrum 9"*' suggesting that such changes are related to specific aspects of differentiation and maturation of nervous tissues both in vivo and in vitro. Although the explants were grown with attached DRG, which were not included in the fresh tissue examined, the types of gangliosides present in DRG are the same as those observed in cord tissues. 13Moreover, since the fresh tissues contained DRG fiber tracts and terminations, the ganglioside differences observed between the cultured and fresh tissues were probably not simply due to the lack of DRG cell bodies in the latter group. The only qualitative differences measured between the two culture groups were the presence of GM3 and GM2 in the galactose-grown explants where they represented 37% of the ganglioside content in these cultures. High levels of these gangliosides are normally associated with glial cells, 8 however, smaller amounts of GM3 and GM2 have been found in pure neuronal cultures, showing that both can be normal constituents of nerve cells as well. 9J2 It is not known whether, or in what respect, the two culture groups may differ in either their neural or their glial content, making it impossible to speculate on the cellular source of either ganglioside based on the present data. However, the appearance of these two compounds appears to be dependent on the presence of galactose in the culturing medium. The fate of the exogenously added galactose is unknown, though our use of galactose-l-phosphate suggests that it may be incorporated locally, at the cell s u r f a c e . 28'32 The restriction of GM3 and GM2 to cultures which exhibit sensory afferent selectivity suggests that these gangliosides may be in some way associated with this phenomenon. GM2 has been suggested as a possible selectivity factor in developing chick retinotectal adhesion, 2~ while Blackburn et al. 4 have shown this ganglioside to be one of the most adhesive of the major gangliosides in the chick neural retina. Although GM2 and GM3 have been shown to occur in immature neuronal tissues in vivo as well as in vitro, their levels decrease with maturation and may even be missing in mature neurons, x This may account for the lack of detectable amounts of these gangliosides in both postnatal and adult cord tissues in the present study. Why GM2 and GM3 are present in vitro but not detected in vivo is unknown. If either or both gangliosides are involved in selectivity, then fresh SC-DRG, which evince selective projection patterns, ought to contain measurable amounts of both. One possible explanation for these differences may be related to the levels of bioelectric activity in the two situations. Hinrichs et al.'5 have shown that increased depolarization of cerebellar neurons in vitro leads to elevated levels of GM1, GM2, GM3, and GD3, but not the major di- and trisialo gangliosides. CDMogrown explants show increasing levels of spontaneous bioelectric discharges with time in vitro, 2 which, in turn, may
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account for the presence of GM2 and GM3 in those cultures grown in the presence of the most appropriate chemical substrates. It was interesting to note that the levels of G D l a , G D l b and G T l b were higher in the CDM controls than in either the galactose-grown group or age-matched fresh tissues. These arc gangliosides presumed to be associated with differentiation and maturation of nervous tissues in vivo and in vitro, particularly with the formation of synapses? ~~ The amounts of these gangliosides in the galactose-grown explants, on the other hand, were similar to the age-matched fresh tissues. These data suggest that differentiation and maturation is occurring in both the m vitro groups, and that those grown in the presence of galactose most closely resemble agematched tissues. Levels of GM1 and GD3 were similar in both groups of cultures. The amount of G M l was less than that found in neonatal (and adult) spinal cord, but this would be expected in an in vitro situation where reduced levels of myelination are occurring. GM1 is a normal constituent of neurons ~2 and it is probable that the amounts measured in the current study represent the G M l present in D R G and spinal nerve cells. The high levels of GD3 in both culture groups may be indicative of increased mitotic activity in the cultures, perhaps associated with glial production. 26.27 The data obtained in the current study has established ganglioside profile patterns for mouse spinal cord at a fetal and postnatal stage of development corresponding to explantation and harvesting of S C - D R G explants in vitro. Since D R G afferents evince a selective preference for dorsal regions of galactose-grown cord explants, regional ganglioside differences may exist in this plane as well as in the rostrocaudai axis. It would now be of interest to examine dorsal and ventral halves of the galactose-grown cord explants and thereby determine whether there are dorsoventral differences in any of the gangliosides present in these cultures which might indicate that these compounds could also be contributing to selective projection patterns as has been suggested for in the visual system. 2~ In addition, if such observations could also be made in SCD R G grown in other serum-free media which do or do not support sensory afferent selectivity, it might be possible to correlate selectivity with the quality or quantity of the gangliosides present in these tissues. A c k n o w l e d g e m e n t s - - T h e authors wish to express their appreciation to Drs M. A. Corner, J. M. Ruijter, M. Mirmiran and H. Romijn for their helpful comments on the manuscript and project. We also thank Mr H. Stoffels for his art work on the two figures and Mr G. van der Meulen for his photographic assistance.
REFERENCES 1. Baker R. E. (1983) Effects of gangliosides on the development of selective afferent connections within fetal mouse spinal cord explants. Neurosci. Lett. 41, 81-84. 2. Baker R. E., Habets A. M. M. C., Brenner E. and Corner M. A. (1982) lnfluence of growth medium, age in vitro and spontaneous bioelectric activity on the distribution of sensory ganglion-evoked activity in spinal cord explants. Devl Brain Res. 5, 329-341. 3. Baker R. E., Corner M. A. and Kleiss M. (1983) Effects of chemical additives on functional innervation patterns in mouse spinal cord-ganglion explants in serum-free medium. Neurosci. Lett. 41,321-324. 4. Blackburn C. C., Swank-Hill P. and Schnaar R. L. 0986) Gangliosides support neural retina cell adhesion. Y. bioL Chem. 261, 2873-2881. 5. Crain S. M. and Peterson E. R. (1967) Onset and development of functional interneuronal connections in explants of rat spinal cord-ganglia during maturation in culture. Brain Res. 6, 750-762. 6. Crain S. M., Bornstein M. B. and Peterson E. R. (1968) Maturation of cultured embryonic CNS tissues during chronic exposure to agents which prevent bioelectric activity. Brain Res. 8, 363-372. 7. Dawson G. and Stefansson K. (1984) Gangliosides of human spinal cord: aberrant composition of cords from patients with amyotrophic lateral sclerosis. J. Neurosci. Res. 12, 213-220. 8. Dreyfus H., Louis J. C.. Harth S. and Mandel P. (1980) Gangliosides in cultured neurons. Neuroscience 5, 1647-1655. 9. Dreyfus H., Harth S., Massarelli R. and Louis J. C. (1981) Mechanisms of differentiation in cultured neurons: involvement of gangliosides. In Gangliosides in Differentiation and Neuromuscular Function (eds Raport M. M. and Gorio A.), pp. 151-170. Raven Press, New York. 10. Dreyfus H., Harth S., Guiliani-Debernardi A., Roos M., Mack G. and Mandel P. (1982) Gangliosides in various brain areas of three inbred strains of mice. Neurochem. Res. 7, 477-488. I I. Dreyfus H., Ferret B., Harth S., Gorio A., Durand M., Freysz L. and Massarelli R. (1984) Metabolism and function of gangliosides in developing neurons. J. Neurosci. Res. 12, 31 i-322. 12. Durand M.. Guerold B., Lombard-Golly D. and Dreyfus H. (1986) Evidence for the effects of gangliosides on the development of neurons in primary cultures. In Gangliosides and Neuroplasticity (eds Tettamanti G., Ledeen R. W., Sandhoff K., Nagai Y. and Toffano G.), pp. 295-307. Liviana Press, Padova, Italy. 13. Graves M., Varon S. and McKhann (3. (1969) The effect of nerve growth factor (N(3F) on the synthesis of gangliosides. J. Neurochem. 16, 1533-1541.
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14. Habets A. M. M. C., Baker R. E., Brenner E. and Corner M. A. (1981) Chemically defined medium enhances bioelectric activity in mouse spinal cord-dorsal root ganglion cultures. Neurosci. Len. 22, 51-56. 15. Hinrichs U., Thomsen S., Van Echten G. and Sandhoff K. (1987) Effect of veratrine on ganglioside biosynthesis in cerebellar cultures. In Gangliosides and Modulation of the Neuronal Functions (ed. Rahmann H.), pp. 319-320. Springer-Verlag, Heidelberg. 16. Kracun i., R6sner H., Cosovic C. and Stravljenic A. (1984) Topographical atlas of the gangliosides of the adult human brain. J. Neurochem. 43, 979-989. 17. Landa C. A. and Moscona A. A. (1985) Changes in ganglioside profile in chick embryo retina: studies on tissue and cell cultures, lnt. J. devl Neurosci. 3, 77-88. 18. Landa C. A., Panzetta P. and Maccioni H. J. F. (1984) Biosynthesis of gangliosides in cultured retina from chick embryos. Devl Brain Res. 14, 83-92. 19. Ledeen R. W. (1985) Gangliosides of the neuron. Trends Neurosci. 8, 169-174. 20. Lowry O. H., Rosebrough N. J., Farr D. A. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. 21. Marchase R. B. (1977) Biochemical investigations of retinotectal adhesive specificity. J. Cell Biol. 75, 237-257. 22. Merat A. and Dickerson J. W. T. (1973) The effect of development on the gangliosides of rat and pig brain. J. Neurochem. 20, 873-880. 23. Miettinen T. and Takki-Luukkainen I. T. (1959) Use of butylacetate in determination of sialic acid. Acta Chem. Scand. 13, 856-858. 24. R6sner H. (1982) Ganglioside changes in the chicken optic lobes as biochemical indicators of brain development and maturation. Brain Res. 236, 49-61. 25. Seybold U. and Rahmann H. (1985) Brain gangliosides in birds with different types of postnatal development (nidifugous and didicolous type). Devl Brain Res. 17, 201-208. 26. Seyfried T. N., Miyazawa N. and Yu R. K. (1983) Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant. J. Neurochem. 41,491-505. 27. Seyfried T. N., Bernard D. J. and Yu R. K. (1984) Cellular distribution of gangliosides in the developing mouse cerebellum: analysis using the staggerer mutant. J. Neurochem. 43, 1152-1162. 28. Shur B. D., Vogler M. and Kosher R. A. (1982) Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis. Expl Cell Res. 137, 229-237. 29. Smalheiser N. R., Peterson E. R. and Crain S. M. (1981) Neurites from mouse retina and dorsal root ganglion explants show specific behavior within co-cultured tectum or spinal cord. Brain Res. 208, 499-505. 30. Svennerholm L. (1957) Quantitative estimation of sialic acids--ll. A colorimetric resorcinol-hydrochloric method. Biochim. Biophys. Acta 24, 604-611. 31. Svennerholm L. (1980) Ganglioside designation. In Structure and Function of Gangliosides (eds Svennerholm L., Mandel P., Dreyfus H. and Urban P. F.), p. 11. Plenum Press, New York. 32. Tolvanen M. and Gahmberg C. (3. (1986) In vitro attachment of mono- and oligosaccharides to surface glycoconjugates of intact cells. J. biol. Chem. 261, 9546-9551. 33. Ueno K., Ando S. and Yu R. K. (1978) Gangliosides of human, cat and rabbit spinal cords and cord myelin. J. Lipid Res. 19, 863-871. 34. Vanier M. T., Holm M., Ohman R. and Svennerholm L. (1971) Developmental profiles of gangliosides in human and rat brain. J. Neurochem. 18, 581-592.
DN 7:I-F