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GM1 ganglioside stimulates the regeneration of dopaminergic neurons in the central nervous system
Brain Research, 261 (1983) 163-166
163
Elsevier Biomedical Press
GM1ganglioside stimulates the regeneration of dopaminergic neurons in the central nervous system G. T O F F A N O , G. SAVOINI, F. MORONI, G. LOMBARDI, L. CALZA and L. F. A G N A T I
Department of Biochemistry, Fidia Research Laboratories, 35031 Abano Terme, (Italy), (F.M., G.L.), Institute of Pharmacology, University of Florence, 50134 Florence, and (L. C., L.F.A.), Institute of Human Physiology, University of Modena, 41100 Modena (Italy) (Accepted September 28th, 1982)
Key words: GM 1ganglioside - nigro-striatal DA neurons - hemitransection - sprouting response - immunohistochemistry
The effect o f G M 1ganglioside on the recovery of nigro-striatal dopaminergic neurons was studied in rats after unilateral hemitransection. We find that repeated administration of GM l significantly increases the HVA content, the tyrosine hydroxylase activity and the tyrosine hydroxylase-related immunofluorescence in the striatum ipsilateral to the lesion. Furthermore, GM] reduces the sensitivity of lesioned rats to apomorphine. The data are compatible with the view that a functional dopaminergic reinnervation of the striatum is facilitated by GM1 treatment after hemitransection.
Although the mechanism governing regenerative growth in the central nervous system is still unknown, many types of central neurons exhibit a pronounced capacity for axon sprouting and formation of new connections after lesions:,L In searching for factors affecting the neurite growth response particular attention has been devoted to gangliosides. Gangliosides, sialic acid-containing glycosphingolipids, are normal components of the m a m m a l i a n plasma membranes and particularly abundant in the nervous tissue t2. Because of their asymmetrical localization in the outer leaflet of membrane bilayer, they are assumed to play a role in cell surface events such as cell-cell recognition 27 and biotransduction of membrane-mediated information 7. Moreover, gangliosides stimulate the morphological and biochemical differentiation of neuronal clonal and primary cell cultures 19, while in vivo they enhance axonal sprouting, during peripheral reinnervation in rat and cat4,~° and accelerate the recovery of choline acetyltransferase and acetylcholinesterase activities in rat hippocampus after medioventral septal lesion26. In the present study we have investigated the effect of the monosialoganglioside GM123 o n the recovery of nigrostriatal dopaminergic neurons after unilateral hemitransection. 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
Sprague-Dawley rats weighing 170-190 g were anesthetized with 3 m l / k g Ketalar (ParkeDavis) and unilaterally hemitransected by means of a 4 mm-wide knife lowered with an angle of 65° at the level of the coronary suture (AP 82, K6nig-Klippel Atlas) as previously reported ~4. This surgical procedure reduces, one week later, by approximately 50% the HVA content (from 82.62 __.4.5 to 39 ___ 3.8 p m o l / m g prot.) and the apparent Vmax of striatal TH for D M P H 4 (from 18.23 _+ 1.02 to 8.69 ___0.81 nmol CO2"h-~ "mg-1 prot.) in the striatum of the lesioned side, determined as we have already described 8. The apparent Km of TH changes from 1.10 ___ 0.07 in the unlesioned to 0.71 -!-_0.04 m M in the lesioned side. This latter change possibly reflects a compensatory mechanism due to the loss of control by the striato-nigral GABA pathways on the nigro-striatal dopaminergic neurons 8,14. The time-course of changes in TH activity in the striatum of the lesioned side shows that in saline-treated animals the Vmaxdecreases, 8 days after surgery, to 50% of that of the contralateral unlesioned side, and it raises by only 10% on day 76 (Fig. 1). In contrast, in animals treated with 30 m g / k g G M 1 there is a significant increase in the Vmax of TH in the lesioned side starting on day 14 (P < 0.01 with respect to saline-treated
164 control unlesioned side
on the TH activity of the unlesioned side, or in unoperated and sham-operated animals up to the longest post-operative time, i.e. for 76 days. The GM~ treatment also does not affect the body weight of both operated and unoperated animals. The increase of TH activity induced by GM~ is associated with a concomitant increase of HVA (Table I). Five mg/kg GM~ caused a significant recovery in the lesioned side already at day 14, which was almost complete at day 45. Thirty mg/kg GM~ produces a complete recovery already at day 14. The sensitivity of rats to apomorphine-induced rotation was significantly reduced by the GM1 treatment. The cycling was ipsiversive, probably because hemitransection produced a decreased density of DA binding sites in the striatum of the lesioned side (manuscript in preparation). Apomorphine (1 mg/kg s.c.) caused in the hemitransected saline-treated rats 340 _ 8 and 425 _+ 18 turns/45 min and in the 5 mg/kg GM~-treated rats 275 _ 1 and 300 _+ 15 turns/45 min 2~ at day 14 and 30, respectively. Finally, using an immunohistochemical technique a significant increase of TH-related immunofluorescence in the striatum ipsilateral to the lesion was observed in animals treated with GM~ with respect to the control group (Fig. 2). The present data indicate that administration of GM~ ganglioside to rats with unilateral hemitransection is followed by an improvement of biochemical, immunohistochemical and behav-
aO, 60~
a¢
20, 0 8
14
45
76
Days after hemitransection
Fig. 1. Time-course of changes in the activity of TH in the striatum ipsilateral to hemitransection: saline (El) or 30 m g / k g i.p. G M l (~) injected. G M l monosialoganglioside was extracted and purified from calf brain. Identification, structural analysis and purity were assessed as previously reported 24. The purity was over 99%. G M l, dissolved in saline, was given intraperitoneally starting on the second day after surgery and finishing 24 h before sacrifice. Data represent Vma x m e a n values of TH expressed in percent of respective unlesioned side used as control. All means are from 6-8 separate determinations. The Vma x of TH determined in the unlesioned side of the saline-treated group (18.73 ± 0.95 nmol CO2.h-l.mg -I prot.) was not significantly different from that of GMl-treated one (19.21 _ 1.03 nmol CO2. h- l 'mg -1 prot.) 8 days after surgery and remained in the same range when measured at day 14, 45 and 76. *P ~ O.Ol with respect to the group injected with saline (Student's t-test).
group). The effect of GM l treatment is dose-dependent (Table I). Fourteen days after surgery, the Vmax of TH in the lesioned side of 5 and 30 mg/kg-treated group is 73 and 85% of that of the unlesioned side, respectively. The increase of Vma~elicited by GM~, is paralleled by an increase of Kin. The ganglioside treatment has no effect TABLE I
Effect of G M l injection on striatal TH activity and H VA content of hemitransected rats Values are means ± S.E. of 6-8 separate determinations done in duplicate. Treatment with G M I started on the second day after surgery and lasted for 12 days. Animals were sacrificed 24 h after the last treatment. Vma x --- nmol CO2.h-t.mg -I prot.; K m = raM; HVA = p m o l / m g prot., determined as previously reported 6.
Treatment
Unlesioned side
Lesioned side
TH
Saline GM l 5 mg/kg GM 130mg/kg
H VA
Vmax
Krn
19.21 _ 0.93 18.76 _ 0.81 21.06 ± 1.03
0.95 - 0.04 1.01 - 0.06 0.92 ± 0.08
80 ± 4 83 - 6 78 ± 7
* P < 0.01 - 0.001 vs the respective unlesioned side. ** P , ( 0.01 - 0.001 vs the lesioned side of saline-treated group.
TH
H VA
V,nax
Km
9.10 ± 0.92* 13.89 ± 0.95*.** 17.90 ± 1.01"*
0.65 - 0.04* 0.78 ± 0.05* 0.83 ± 0.03**
36 - 6* 62 ± 5",** 80 ± 8**
165
Fig. 2. TH-positive terminals in the striatum (A 7600, K6nig-Klippel atlas). The rats were anesthetized and perfused with 4% paraformaldehyde solution and the brains were transversely cut in a cryostat. The sections were 14/tm thick and in the immunohistochemical procedure the TH antiserum was used in a dilution of 1:200 containing 0.3% Triton X- 100 II. Micrographs of the immunoreactive terminals were taken by means of Triton X-100 films. Upper panel on the left, the intact side of a control animal. Upper panel on the right, the lesioned side of a control animal. Lower panel on the left, the intact side of an animal treated with GM 1(10 mg/kg i.p. for 14 days). Lower panel on the fight, the lesioned side of an animal treated with GM~ (10 mg/kg i.p. for 14 days).
ioral parameters which define the nigro-striatal dopaminergic pathways. The GM~ treatment is accompanied by a dose-dependent increase of TH activity and HVA content in the striatum ipsilateral to the lesion, while no changes can be detected in the unlesioned side of operated, in unoperated or sham-operated animals. This suggests that GM~ ganglioside does not influen1 Benfey, M. and Aguayo, A. J., Extensive elongation of axons from rat brain into peripheral nerve grafts, Nature (Lond.), 296 (1982) 150-152. 2 BjOrklund, A. and Stenevi, U., Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system, PhysioL Rev., 59 (1979) 62-100. 3 Bj6rklund, A. and Stenevi, U., In vivo evidence for a hip-
ce per se the striatal TH activity or the HVA content. Moreover, ganglioside treatment increases the TH-related immunofluorescence in the striatum ipsilateral to the lesion and reduces the sensitivity of lesioned rats to apomorhine. The data are compatible with the view that a functional dopaminergic reinnervation of the striatum is facilitated by GMt treatment after hemitransection. Collateral sprouting of mesolimbic and nigro-striatal dopaminergic neurons has been observed after axonal electrolytic injury9,2°. Spontaneous sprouting of some nerve fibers can be elicited by tropic factors unmasked by lesion3 and specific neurite growth-promoting factors for mesencephalic dopaminergic neurons can be found in striatal membranes 17. A possible correlation between trophic factors and gangliosides has been suggested22. A relationship seems to exist between GM1 appearance and neurite outgrowth in the cerebellum during development25 while mature brains afflicted by GMI gangliosidosis develop meganeurites with secondary extensions 18. Furthermore, GM~ crosses the brainblood barrier ~5and in vitro interacts with neuronal membranes24 modifying the (Na÷,K+)AT Pase ~3and adenylate cyclase 16activities. In conclusion, these data indicate that GM~ injection results in an increase of central dopaminergic axonal sprouting either by facilitating the action of neurite elongation or sprouting factors or by removing factors which inhibit the nerve fiber regeneration I. The source of regrowing dopaminergic nerve terminals in the striatum remains to be investigated. Possible candidates could be the damaged fibers, the remaining intact axons or dopaminergic neurons originated from other compartments. Mass-spectrometric analyses of HVA were performed at Mass-Spectrometry Center, Medical School, University of Florence, Italy. pocampal adrenergic neuronotrophic factor specifically released on septal deafferentation, Brain Research, 229 (1981) 403-428. 4 Ceccarelli, B., Aporti, F. and Finesso, M., Effects of brain gangliosides on functional recovery in experimental regeneration and reinnervation, Advanc. Exp. Med. Biol., 21 (1976) 275-293.
166 5 Cotman, C. W., Nieto-Sampedro, M. and Harris, E. W., Synapse replacement in the nervous system of adult vertebrates, Physiol. Rev., 61 ( 1981 ) 684--784. 6 Dziedzic, S. W., Bertani-Dziedzic, L. and Gitlow, S. E., Separation and determination of urinary homovanillic acid and iso-homovanillic acid by gas-liquid chromatography and electron capture detection. J. lab. Clin. Meal, 82 (1973) 829-835. 7 Fishman, P. H. and Brady, R. O., Biosynthesis and function ofgangliosides, Science, 194 (1976) 906-915. 8 Gale, K., Costa, E., Toffano, G., Hong, J. S. and Guidotti, A., Evidence for a role of nigral y-aminobutyric acid and substance P in the haloperidol-induced activation of striatal lyrosine hydroxylase, J. Pharmacol. exp. Ther., 206(1978)29 37. 9 Gilad, G. M., and Reis, D. J., Collateral sprouting in cerebral mesolimbic dopamine neurones: biochemical and immunocytochemical evidence of changes in the activity and distribution of tyrosine hydroxylase in terminal fields and in cell bodies, Brain Research, 160 (1979) 1736. 10 Gorio, A., Carmignoto, G., Facci, L. and Finesso, M., Motor sprouting induced by ganglioside treatment. Possible implication for gangliosides on neuronal growth, Brain Research, 197 (1980) 236-241. 11 HOkfelt, T., Fuxe, K., Goldstein, M. and Joh, T. H., Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology, Histochemie, 33 (1973) 231-254. 12 Ledeen, R. W., Ganglioside structures and distribution: are they localized at the nerve ending?, J. Supramol. Struct., 8 (1978) 1- 17. 13 Leon, A., Facci, L., Toffano, G., Sonnino, S. and Tettamanti, G., Activation of (Na +,K +)ATPase by nanomolar concentrations o f G M 1ganglioside, J. Neurochem., 37 ( 198 I) 350--357. 14 Mao, C. C., Peralta, E., Moroni, F. and Costa, E., The turnover rate of ~,-aminobutyric acid in the substantia nigra following electrical stimulation or lesioning of the striatonigral pathways, Brain Research, 155 (1978) 147 152. 15 Orlando, P., Cocciante, G., Ippolito, G., Massari, P., Roberti, S. and Tettamanti, G., The fate of tritium labeled GM I ganglioside injected in mice, Pharmacol. Res. Commun., 11 (1979)759 773. 16 Partington, C. R. and Daly, J. M., Effect ofgangliosides on adenylate cyclase activity in rat cerebral cortical
membranes, Molec. Pharmacol., 15 (1979) 484-491. 17 Prochiantz, A., Daguet, M. C., Herbet, A. and Glowinski, J., Specific stimulation of in vitro maturation of mesencephalic dopaminergic neurones by striatal membranes, Nature (Lond.), 293 ( 1981) 570-572. 18 Purpura, D. P., Pappas, G. D. and Baker, H. J., Meganeurites and other aberrant processes of neurones on GM l-gangliosidosis. A Golgi study, Brain Research, 145 (1977) 13-26. 19 Rapport, M. M. and Gorio, A. (Eds.), Gangliosides in Neurological and Neuromuscular Function, Development andRepair, Raven Press, New York, 1981. 20 Reis, D. J., Gilad, G., Pickel, V. M. and Joh, T. H., Reversible changes in the activities and amounts of tyrosine hydroxylase in dopamine neurons of the substantia nigra in response to axonal injury as studied by immunochemical and immunocytochemical methods, Brain Research, 144 (1978)325 342. 21 Schwarcz, R., Fuxe, K., Agnati, L. F., HOkfelt, T. and Coyle, J. T., Rotational behaviour in rats with unilateral striatal kainic acid lesions: a behavioural model for studies on intact dopamine receptors, Brain Research, 170 (1979) 485-495. 22 Seifert, W., Gangliosides in nerve cell cultures. In M. M. Rapport and A. Gorio (Eds.), Gangliosides in Neurological and Neuromuscular Function, Development and Repair, Raven Press, New York, 1981, pp. 99 - 117. 23 Svennerholm, L., Gangliosides and synaptic transmission. In L. Svennerholm, P. Mandel, H. Dreyfus and P. F. Urban (Eds.), Structure and Function of Gangliosides, Plenum Press, New York, 1980, pp. 533 544. 24 Toffano, G., Benvegnfi, D., Bonetti, A. C., Facci, L., Leon, A., Orlando, P., Ghidoni, R. and Tettamanti, G., Interactions of GMI ganglioside with crude rat brain neuronal membranes, J. Neurochem., 35 (1980) 861-866. 25 Willinger, M. and Schachner, M., GMI ganglioside as a marker for neuronal differentiation in mouse cerebellum, Develop. Biol., 74(1980) 101 107. 26 Wojcik, M., Ulas, J. and Oderfeld-Nowak, B., The stimulating effect of ganglioside injections on the recovery of choline acetyltransferase and acetylcholinesterase activities in the hippocampus of the rat after septal lesions, Neurosci., 7 (1982) 495--499. 27 Yamakawa, T. and Nagai, Y., Glycolipids at the cell surface and their biological functions, TIBS 3 (1978) 128131.
163
Elsevier Biomedical Press
GM1ganglioside stimulates the regeneration of dopaminergic neurons in the central nervous system G. T O F F A N O , G. SAVOINI, F. MORONI, G. LOMBARDI, L. CALZA and L. F. A G N A T I
Department of Biochemistry, Fidia Research Laboratories, 35031 Abano Terme, (Italy), (F.M., G.L.), Institute of Pharmacology, University of Florence, 50134 Florence, and (L. C., L.F.A.), Institute of Human Physiology, University of Modena, 41100 Modena (Italy) (Accepted September 28th, 1982)
Key words: GM 1ganglioside - nigro-striatal DA neurons - hemitransection - sprouting response - immunohistochemistry
The effect o f G M 1ganglioside on the recovery of nigro-striatal dopaminergic neurons was studied in rats after unilateral hemitransection. We find that repeated administration of GM l significantly increases the HVA content, the tyrosine hydroxylase activity and the tyrosine hydroxylase-related immunofluorescence in the striatum ipsilateral to the lesion. Furthermore, GM] reduces the sensitivity of lesioned rats to apomorphine. The data are compatible with the view that a functional dopaminergic reinnervation of the striatum is facilitated by GM1 treatment after hemitransection.
Although the mechanism governing regenerative growth in the central nervous system is still unknown, many types of central neurons exhibit a pronounced capacity for axon sprouting and formation of new connections after lesions:,L In searching for factors affecting the neurite growth response particular attention has been devoted to gangliosides. Gangliosides, sialic acid-containing glycosphingolipids, are normal components of the m a m m a l i a n plasma membranes and particularly abundant in the nervous tissue t2. Because of their asymmetrical localization in the outer leaflet of membrane bilayer, they are assumed to play a role in cell surface events such as cell-cell recognition 27 and biotransduction of membrane-mediated information 7. Moreover, gangliosides stimulate the morphological and biochemical differentiation of neuronal clonal and primary cell cultures 19, while in vivo they enhance axonal sprouting, during peripheral reinnervation in rat and cat4,~° and accelerate the recovery of choline acetyltransferase and acetylcholinesterase activities in rat hippocampus after medioventral septal lesion26. In the present study we have investigated the effect of the monosialoganglioside GM123 o n the recovery of nigrostriatal dopaminergic neurons after unilateral hemitransection. 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
Sprague-Dawley rats weighing 170-190 g were anesthetized with 3 m l / k g Ketalar (ParkeDavis) and unilaterally hemitransected by means of a 4 mm-wide knife lowered with an angle of 65° at the level of the coronary suture (AP 82, K6nig-Klippel Atlas) as previously reported ~4. This surgical procedure reduces, one week later, by approximately 50% the HVA content (from 82.62 __.4.5 to 39 ___ 3.8 p m o l / m g prot.) and the apparent Vmax of striatal TH for D M P H 4 (from 18.23 _+ 1.02 to 8.69 ___0.81 nmol CO2"h-~ "mg-1 prot.) in the striatum of the lesioned side, determined as we have already described 8. The apparent Km of TH changes from 1.10 ___ 0.07 in the unlesioned to 0.71 -!-_0.04 m M in the lesioned side. This latter change possibly reflects a compensatory mechanism due to the loss of control by the striato-nigral GABA pathways on the nigro-striatal dopaminergic neurons 8,14. The time-course of changes in TH activity in the striatum of the lesioned side shows that in saline-treated animals the Vmaxdecreases, 8 days after surgery, to 50% of that of the contralateral unlesioned side, and it raises by only 10% on day 76 (Fig. 1). In contrast, in animals treated with 30 m g / k g G M 1 there is a significant increase in the Vmax of TH in the lesioned side starting on day 14 (P < 0.01 with respect to saline-treated
164 control unlesioned side
on the TH activity of the unlesioned side, or in unoperated and sham-operated animals up to the longest post-operative time, i.e. for 76 days. The GM~ treatment also does not affect the body weight of both operated and unoperated animals. The increase of TH activity induced by GM~ is associated with a concomitant increase of HVA (Table I). Five mg/kg GM~ caused a significant recovery in the lesioned side already at day 14, which was almost complete at day 45. Thirty mg/kg GM~ produces a complete recovery already at day 14. The sensitivity of rats to apomorphine-induced rotation was significantly reduced by the GM1 treatment. The cycling was ipsiversive, probably because hemitransection produced a decreased density of DA binding sites in the striatum of the lesioned side (manuscript in preparation). Apomorphine (1 mg/kg s.c.) caused in the hemitransected saline-treated rats 340 _ 8 and 425 _+ 18 turns/45 min and in the 5 mg/kg GM~-treated rats 275 _ 1 and 300 _+ 15 turns/45 min 2~ at day 14 and 30, respectively. Finally, using an immunohistochemical technique a significant increase of TH-related immunofluorescence in the striatum ipsilateral to the lesion was observed in animals treated with GM~ with respect to the control group (Fig. 2). The present data indicate that administration of GM~ ganglioside to rats with unilateral hemitransection is followed by an improvement of biochemical, immunohistochemical and behav-
aO, 60~
a¢
20, 0 8
14
45
76
Days after hemitransection
Fig. 1. Time-course of changes in the activity of TH in the striatum ipsilateral to hemitransection: saline (El) or 30 m g / k g i.p. G M l (~) injected. G M l monosialoganglioside was extracted and purified from calf brain. Identification, structural analysis and purity were assessed as previously reported 24. The purity was over 99%. G M l, dissolved in saline, was given intraperitoneally starting on the second day after surgery and finishing 24 h before sacrifice. Data represent Vma x m e a n values of TH expressed in percent of respective unlesioned side used as control. All means are from 6-8 separate determinations. The Vma x of TH determined in the unlesioned side of the saline-treated group (18.73 ± 0.95 nmol CO2.h-l.mg -I prot.) was not significantly different from that of GMl-treated one (19.21 _ 1.03 nmol CO2. h- l 'mg -1 prot.) 8 days after surgery and remained in the same range when measured at day 14, 45 and 76. *P ~ O.Ol with respect to the group injected with saline (Student's t-test).
group). The effect of GM l treatment is dose-dependent (Table I). Fourteen days after surgery, the Vmax of TH in the lesioned side of 5 and 30 mg/kg-treated group is 73 and 85% of that of the unlesioned side, respectively. The increase of Vma~elicited by GM~, is paralleled by an increase of Kin. The ganglioside treatment has no effect TABLE I
Effect of G M l injection on striatal TH activity and H VA content of hemitransected rats Values are means ± S.E. of 6-8 separate determinations done in duplicate. Treatment with G M I started on the second day after surgery and lasted for 12 days. Animals were sacrificed 24 h after the last treatment. Vma x --- nmol CO2.h-t.mg -I prot.; K m = raM; HVA = p m o l / m g prot., determined as previously reported 6.
Treatment
Unlesioned side
Lesioned side
TH
Saline GM l 5 mg/kg GM 130mg/kg
H VA
Vmax
Krn
19.21 _ 0.93 18.76 _ 0.81 21.06 ± 1.03
0.95 - 0.04 1.01 - 0.06 0.92 ± 0.08
80 ± 4 83 - 6 78 ± 7
* P < 0.01 - 0.001 vs the respective unlesioned side. ** P , ( 0.01 - 0.001 vs the lesioned side of saline-treated group.
TH
H VA
V,nax
Km
9.10 ± 0.92* 13.89 ± 0.95*.** 17.90 ± 1.01"*
0.65 - 0.04* 0.78 ± 0.05* 0.83 ± 0.03**
36 - 6* 62 ± 5",** 80 ± 8**
165
Fig. 2. TH-positive terminals in the striatum (A 7600, K6nig-Klippel atlas). The rats were anesthetized and perfused with 4% paraformaldehyde solution and the brains were transversely cut in a cryostat. The sections were 14/tm thick and in the immunohistochemical procedure the TH antiserum was used in a dilution of 1:200 containing 0.3% Triton X- 100 II. Micrographs of the immunoreactive terminals were taken by means of Triton X-100 films. Upper panel on the left, the intact side of a control animal. Upper panel on the right, the lesioned side of a control animal. Lower panel on the left, the intact side of an animal treated with GM 1(10 mg/kg i.p. for 14 days). Lower panel on the fight, the lesioned side of an animal treated with GM~ (10 mg/kg i.p. for 14 days).
ioral parameters which define the nigro-striatal dopaminergic pathways. The GM~ treatment is accompanied by a dose-dependent increase of TH activity and HVA content in the striatum ipsilateral to the lesion, while no changes can be detected in the unlesioned side of operated, in unoperated or sham-operated animals. This suggests that GM~ ganglioside does not influen1 Benfey, M. and Aguayo, A. J., Extensive elongation of axons from rat brain into peripheral nerve grafts, Nature (Lond.), 296 (1982) 150-152. 2 BjOrklund, A. and Stenevi, U., Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system, PhysioL Rev., 59 (1979) 62-100. 3 Bj6rklund, A. and Stenevi, U., In vivo evidence for a hip-
ce per se the striatal TH activity or the HVA content. Moreover, ganglioside treatment increases the TH-related immunofluorescence in the striatum ipsilateral to the lesion and reduces the sensitivity of lesioned rats to apomorhine. The data are compatible with the view that a functional dopaminergic reinnervation of the striatum is facilitated by GMt treatment after hemitransection. Collateral sprouting of mesolimbic and nigro-striatal dopaminergic neurons has been observed after axonal electrolytic injury9,2°. Spontaneous sprouting of some nerve fibers can be elicited by tropic factors unmasked by lesion3 and specific neurite growth-promoting factors for mesencephalic dopaminergic neurons can be found in striatal membranes 17. A possible correlation between trophic factors and gangliosides has been suggested22. A relationship seems to exist between GM1 appearance and neurite outgrowth in the cerebellum during development25 while mature brains afflicted by GMI gangliosidosis develop meganeurites with secondary extensions 18. Furthermore, GM~ crosses the brainblood barrier ~5and in vitro interacts with neuronal membranes24 modifying the (Na÷,K+)AT Pase ~3and adenylate cyclase 16activities. In conclusion, these data indicate that GM~ injection results in an increase of central dopaminergic axonal sprouting either by facilitating the action of neurite elongation or sprouting factors or by removing factors which inhibit the nerve fiber regeneration I. The source of regrowing dopaminergic nerve terminals in the striatum remains to be investigated. Possible candidates could be the damaged fibers, the remaining intact axons or dopaminergic neurons originated from other compartments. Mass-spectrometric analyses of HVA were performed at Mass-Spectrometry Center, Medical School, University of Florence, Italy. pocampal adrenergic neuronotrophic factor specifically released on septal deafferentation, Brain Research, 229 (1981) 403-428. 4 Ceccarelli, B., Aporti, F. and Finesso, M., Effects of brain gangliosides on functional recovery in experimental regeneration and reinnervation, Advanc. Exp. Med. Biol., 21 (1976) 275-293.
166 5 Cotman, C. W., Nieto-Sampedro, M. and Harris, E. W., Synapse replacement in the nervous system of adult vertebrates, Physiol. Rev., 61 ( 1981 ) 684--784. 6 Dziedzic, S. W., Bertani-Dziedzic, L. and Gitlow, S. E., Separation and determination of urinary homovanillic acid and iso-homovanillic acid by gas-liquid chromatography and electron capture detection. J. lab. Clin. Meal, 82 (1973) 829-835. 7 Fishman, P. H. and Brady, R. O., Biosynthesis and function ofgangliosides, Science, 194 (1976) 906-915. 8 Gale, K., Costa, E., Toffano, G., Hong, J. S. and Guidotti, A., Evidence for a role of nigral y-aminobutyric acid and substance P in the haloperidol-induced activation of striatal lyrosine hydroxylase, J. Pharmacol. exp. Ther., 206(1978)29 37. 9 Gilad, G. M., and Reis, D. J., Collateral sprouting in cerebral mesolimbic dopamine neurones: biochemical and immunocytochemical evidence of changes in the activity and distribution of tyrosine hydroxylase in terminal fields and in cell bodies, Brain Research, 160 (1979) 1736. 10 Gorio, A., Carmignoto, G., Facci, L. and Finesso, M., Motor sprouting induced by ganglioside treatment. Possible implication for gangliosides on neuronal growth, Brain Research, 197 (1980) 236-241. 11 HOkfelt, T., Fuxe, K., Goldstein, M. and Joh, T. H., Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology, Histochemie, 33 (1973) 231-254. 12 Ledeen, R. W., Ganglioside structures and distribution: are they localized at the nerve ending?, J. Supramol. Struct., 8 (1978) 1- 17. 13 Leon, A., Facci, L., Toffano, G., Sonnino, S. and Tettamanti, G., Activation of (Na +,K +)ATPase by nanomolar concentrations o f G M 1ganglioside, J. Neurochem., 37 ( 198 I) 350--357. 14 Mao, C. C., Peralta, E., Moroni, F. and Costa, E., The turnover rate of ~,-aminobutyric acid in the substantia nigra following electrical stimulation or lesioning of the striatonigral pathways, Brain Research, 155 (1978) 147 152. 15 Orlando, P., Cocciante, G., Ippolito, G., Massari, P., Roberti, S. and Tettamanti, G., The fate of tritium labeled GM I ganglioside injected in mice, Pharmacol. Res. Commun., 11 (1979)759 773. 16 Partington, C. R. and Daly, J. M., Effect ofgangliosides on adenylate cyclase activity in rat cerebral cortical
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