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Specific lysis by 6-hydroxydopamine of catecholaminergic synaptosomes prepared from rat cerebrum
374
Brain Re.search. 321 (1984) 374 370 Elscvwr
BRE 20438
Specific lysis by 6-hydroxydopamine of catecholaminergic synaptosomes prepared from rat cerebrum TATSUO SUZUKI, KUNIKO OKUMURA-NOJ1 and RYO TANAKA Department of Biochemistry, Nagoya City University Medical School, Mizuho-Ku, Nagoya 407 (Japan)
(Accepted June 19th, 1984) Key words 6-hydroxydopamine - - catecholaminergic synaptosome - - synaptolysis - - rat cerebrum
The toxicity of 6-hydroxydopamine on synaptosomes prepared from rat cerebra was investigated. The drug caused synaptolysis, and the lytic effect was specific to catecholaminergic synaptosomes, since no neurotransmitters (serotonin and amino acids) other than catecholamines (dopamine and noradrenaline) were liberated by the drug. The specific toxicity of 6-hydroxydopamine ( 6 - O H D A ) to catecholaminergic neurons has been well established in vivo and with cultured cells 1-3,14.16~17,19,20. The specificity is believed to be determined by either the selective uptake of the drug 9,1°.15 or the weak activity of the radical elimination in catecholaminergic neurons 18. We examined the specificity of 6 - O H D A toxicity on synaptosomes prepared from rat cerebra. Dopamine, 6-OHDA, 5-hydroxytryptamine (5-HT), and 3,4-dihydroxybenzylamine ( D H B A ) were obtained from Sigma Chemical Co. ; noradrenaline and pargyline hydrochloride from Katayama Chemical Co.; Ficoll from Pharmacia Fine Chemicals; Amberlite CG-50 from R o h m and Hass; and nicotinamide adenine dinucleotide from Wako Pure Chemical Industries, Ltd. Synaptosomes were prepared from the cerebra of Wistar rats (6 weeks old, male) by the method of Cotman and Matthews a. The synaptosomes collected from a 7 . 5 - 1 3 % Ficoll interface were suspended in a physiological salineS, incubated at 0 °C for 15-30 min, spun down at 10,000 g for 5 min, and resuspended in the saline to make a final protein concentration about 9 mg/ml. The treatment of synaptosomes with 6 - O H D A was started by the addition of 6 - O H D A to 100 ~1,100~1, 200 ~1, or 400/~1 of synaptosomal suspensions for lac-
tate dehydrogenase ( L D H ) , amino acid neurotransmitter, catecholamine, or 5-HT liberation tests, respectively. After incubation at 30 °C for the periods of time indicated synaptosomes were spun down at 10,000 g for 5 min. The supernatant was used for L D H , catecholamine, 5-HT, or amino acid assay. Catecholamines and 5-HT were determined by a modification of the method of Nagatsu et al. 13. To the synaptosomal supernatant 5 ng of D H B A as internal standard, 20 yl of 1 N perchloric acid, 10 ~tl of 20 mM ethylenediaminetetraacetic acid, and 10 gl of 10% Na/S204 were added, and the denatured protein was removed by centrifugation at 2000 g for 5 rain. The clear supernatant obtained after pH adjustment to 6.1-6.3 with 5 M K2CO 3 and centrifugation at 2000 g for 5 min, was passed through an Amberlite CG-50 column (0.2 ml). The column was washed twice with 1 ml of H20 and once with 0.25 ml of 0.5 M HCI. Amines were eluted with 0.5 ml of 0.5 M HCI and analyzed by high-performance liquid chromatography (Yanaco, ODS-T column) with a voltammetry detector (Yanaco, VMD-101 A). The mobile phases were 0.1 M potassium phosphate buffer for catecholamines and the buffer containing 10%. methanol for 5-HT. The flow rate was 0.8 ml/min and the detector potential was set at 0.8 V against the Ag/AgCI electrode. Amino acid neurotransmitters were assayed by the
Correspondence: T. Suzuki, Department of Biochemistry, Nagoya City University Medical School, Mizuho-Ku, Nagoya 467, Japan.
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
375 method of Dodd et al. 7. To the synaptosomal supernatant, 5 nmol of glucosaminic acid as internal standard and 0.2 N perchloric acid were added, and the protein precipitated was removed by centrifugation at 2000 g for 5 min. Amino acids in the supernatant were analyzed by amino acid analyzer. LDH was assayed by the method of Kornberg 11. Proteins were determined by the method of Lowry et al. 12with bovine serum albumin as standard. Synaptolysis was followed by the determination of LDH liberated from synaptosomes into the incubation medium during 6-OHDA treatment. L D H activity was higher in the medium of 6 - O H D A treatment than in that of the control (data not shown), suggesting that the treatment induced synaptolysis. The specificity of 6 - O H D A toxicity was examined by the assay of catecholamines, 5-HT, and amino acid neurotransmitters liberated from synaptosomes. Large amounts of catecholamines were liberated by 6-OHDA as shown in Fig. 1. The time courses of the catecholamine liberation were similar to that of L D H liberation (not shown), rising immediately from the 0-min incubation and reaching a plateau in 30 min. The amounts of dopamine and noradrenaline liberated after 30 min incubation were 72% and 25%, respectively, of the total amounts contained in synapto-
a
f
.a o
b
TABLE I
Amino acid contents in the cerebral synaptosomes The values were average of the triplicate experiments.
Amino acid
Contents (nmol/g wet wt. of cerebrum)
Tau* Asp* Thr* Ser* Glu* Pro Gly* Ala* Val Met Ile Leu Tyr Phe Lys His Arg GABA*
227 423 37.9 58.0 444 35.6 64.6 52.7 7.5 6.3 9.0 14.3 7.1 31.2 20.6 7.9 20.8 184
* Putative amino acid neurotransmitters.
somes. The amounts of 5-HT liberated by 6 - O H D A were the same as those of the controls (Fig. lb), and were less than 10% of the total in synaptosomes. The amino acid contents of synaptosomes are shown in Table I. Amino acid neurotransmitters, especially taurine, aspartic acid, glutamic acid, and G A B A , were abundant in synaptosomes. The liberation of these amino acids were examined in the presence or absence of 150/zM 6-OHDA (Fig. 2). AI-
100 (%) lOO
i
= 0
u
;2o ~g
.E
0
10
~'eTa u
70 .
20
30
tn~ 0
10
30 60
Glu
..A--'"
20
AI~
201
0
a,~ "~
F
Incubation time (min)
Fig. 1. Liberation of catecholamines and 5-HT from synaptosomes by 6-OHDA. The amounts of catecholamines and 5-HT liberated into the media were assayed in triplicate by high-performance liquid chromatograph with voltammetry. For the detailed procedures see the text. a: catecholamine liberation from synaptosomes by treatment with 150/zM 6 - O H D A at 30 °C. b: liberation of 5-HT by treatment at 30 °C. ~ , dopamine liberated by 6 - O H D A ; - - O - - , dopamine (control); --& , noradrenaline liberated by 6-OHDA; - - A - -, noradrenaline (control); • , 5-HT liberated by 6-OHDA; - - [] - -, 5-HT (control).
_=
s_
~__
GA.A
1 ~0 O.
10
20
30 Incubation
0
0
,
,
,
10
20
30
Time (min)
Fig. 2. Liberation of amino acid neurotransmitters from synaptosomes by 6 - O H D A . The liberation of amino acid neurotransmitters by 6 - O H D A (150¢tM) at 30 °C into the incubation media were assayed in triplicate by an amino acid analyzer. For the detailed procedures see the text. , 6-OHDAtreated; . . . . , control.
376 though the amino acid liberation increased during the
to the transmitter contained 5,~,. Large amounts of do-
incubation, the difference in contents between 6 - O H D A treatment and the control was insignificant. The amounts of amino acids liberated by 6O H D A after 30 rain were 2.4%, 1.4%, 1.0%, 3.2%, 0.3%, 2.8%, 2.4%, and 2.7% of the total contents in the case of taurine, glutamic acid, aspartic acid, threonine, G A B A , alanine, serine and glycine, re-
pamine and noradrenaline together with LDH were liberated from synaptosomes during incubation in the presence of the drug, whereas neither 5-HT nor amino acid neurotransmitters escaped from the synaptosomes. The results indicated that catecholaminergic synaptosomes were destroyed by 6OHDA.
spectively. The present work demonstrates 6 - O H D A toxicity
The present investigation was supported in part by
is specific to the catecholaminergic synaptosomes prepared from rat cerebra. The synaptosomes pre-
the Grant-in-Aid for Scientific Research from the Japanese Ministry of Education.
pared from the brain are heterogeneous with respect
1 Angeletti, P. U. and Levi-Montalcini, R. Sympathetic nerve cell destruction in newborn mammals by 6-hydroxydopamine, Proc. nat. Acad. Sci. U.S.A., 65 (1970) 114-121. 2 Angeletti, P. U. and Levi-Montalcini, R., Cytolytic effect of 6-hydroxydopamine on neuroblastoma cells, Cancer Res., 30 (1970) 2863-2869. 3 Burnstock, G., Morphological changes produced by drugs acting on the autonomic nervous system, Pharmacol. Ther., 5 (1979) 49-53. 4 Cotman, C. W. and Matthews, D. A., Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization, Biochim. biophys. Acta., 249 (1971) 380-394. 5 Docherty, M., Bradford, H. and Anderton, B., Lysis of cholinergic synaptosomes by an antiserum to choline acetyltransferase, FEBS Lett., 144 (1982) 47-50. 6 Docherty, M., Bradford, H. F., Anderton, B. and Wu, JY., Specificlysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase, FEBS Lett., 152 (1983) 57-61. 7 Dodd, P. R., Hardy, J. A., Oakley, A. E., Edwardson, J. A., Perry, E. K. and Delaunoy, J.-P., A rapid method for preparing synaptosomes: comparison with alternative procedures, Brain Research, 226 (1981) 107-118. 8 Fried, R. C. and Blaustein, M. P., Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes), J. Cell Biol., 78 (1978) 685-700. 9 Heikilla, R. and Cohen, G., Inhibition of biogenic amine uptake by hydrogen peroxide: a mechanism for toxic effects of 6-hydroxydopamine, Science, 172 (1971) 1257-1258. 10 Jonsson, G. and Sachs, C., Effects of 6-hydroxydopamine on the uptake and storage of noradrenaline in sympathetic adrenergic neurons, Europ. J. Pharmacol., 9 (1970)
141-155. 11 Kornberg, A., Methods in Enzymology. 1, Academic Press, New York, 1955, pp. 441-443. 12 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 13 Nagatsu, T., Oka, K. and Kato, T., Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography, J. Chromatogr., 163 (1979) 247-252. 14 Prasad, K. N., Effect of dopamine and 6-hydroxydopamine on mouse neuroblastoma cells in vitro, Cancer Res., 31 (1971) 1457-1460. 15 Sachs, C., Jonsson, G., Heikkila, R. and Cohen, G., Control of the neurotoxicity of 6-hydroxydopamine by intraneuronal noradrenaline in rat iris, Acta physiol, scand., 93 (1975) 345-351. 16 Thoenen, H. and Tranzer, J. P., Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine, Naunyn-Schmiedebergs Arch. Pharmacol., 261 (1968) 271-288. 17 Tiffany-Castiglioni, E. and Perez-Polo, J. R., Evaluation of methods for determining 6-hydroxydopamine cytotoxicity, In Vitro, 16 (1980) 59t-599. 18 Tiffany-Castiglioni, E., Saneto, R. P., Proctor, P. H. and Perez-Polo, J. R., Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line, Biochem. Pharmacol., 31 (1982) 181-188. 19 Tiffany-Castiglioni, E. and Perez-Polo, J. R., The role of nerve growth factor in vitro in cell resistence to 6-hydroxydopamine toxicity, Exp. CellRes., 121 (1979) 179-189. 20 Tranzer, J. P. and Thoenen, H., An electron microscopic study of selective, acute degeneration of sympathetic nerve terminals after administration of 6-hydroxydopamine. Experientia, 24 (1968) 155-156.
Brain Re.search. 321 (1984) 374 370 Elscvwr
BRE 20438
Specific lysis by 6-hydroxydopamine of catecholaminergic synaptosomes prepared from rat cerebrum TATSUO SUZUKI, KUNIKO OKUMURA-NOJ1 and RYO TANAKA Department of Biochemistry, Nagoya City University Medical School, Mizuho-Ku, Nagoya 407 (Japan)
(Accepted June 19th, 1984) Key words 6-hydroxydopamine - - catecholaminergic synaptosome - - synaptolysis - - rat cerebrum
The toxicity of 6-hydroxydopamine on synaptosomes prepared from rat cerebra was investigated. The drug caused synaptolysis, and the lytic effect was specific to catecholaminergic synaptosomes, since no neurotransmitters (serotonin and amino acids) other than catecholamines (dopamine and noradrenaline) were liberated by the drug. The specific toxicity of 6-hydroxydopamine ( 6 - O H D A ) to catecholaminergic neurons has been well established in vivo and with cultured cells 1-3,14.16~17,19,20. The specificity is believed to be determined by either the selective uptake of the drug 9,1°.15 or the weak activity of the radical elimination in catecholaminergic neurons 18. We examined the specificity of 6 - O H D A toxicity on synaptosomes prepared from rat cerebra. Dopamine, 6-OHDA, 5-hydroxytryptamine (5-HT), and 3,4-dihydroxybenzylamine ( D H B A ) were obtained from Sigma Chemical Co. ; noradrenaline and pargyline hydrochloride from Katayama Chemical Co.; Ficoll from Pharmacia Fine Chemicals; Amberlite CG-50 from R o h m and Hass; and nicotinamide adenine dinucleotide from Wako Pure Chemical Industries, Ltd. Synaptosomes were prepared from the cerebra of Wistar rats (6 weeks old, male) by the method of Cotman and Matthews a. The synaptosomes collected from a 7 . 5 - 1 3 % Ficoll interface were suspended in a physiological salineS, incubated at 0 °C for 15-30 min, spun down at 10,000 g for 5 min, and resuspended in the saline to make a final protein concentration about 9 mg/ml. The treatment of synaptosomes with 6 - O H D A was started by the addition of 6 - O H D A to 100 ~1,100~1, 200 ~1, or 400/~1 of synaptosomal suspensions for lac-
tate dehydrogenase ( L D H ) , amino acid neurotransmitter, catecholamine, or 5-HT liberation tests, respectively. After incubation at 30 °C for the periods of time indicated synaptosomes were spun down at 10,000 g for 5 min. The supernatant was used for L D H , catecholamine, 5-HT, or amino acid assay. Catecholamines and 5-HT were determined by a modification of the method of Nagatsu et al. 13. To the synaptosomal supernatant 5 ng of D H B A as internal standard, 20 yl of 1 N perchloric acid, 10 ~tl of 20 mM ethylenediaminetetraacetic acid, and 10 gl of 10% Na/S204 were added, and the denatured protein was removed by centrifugation at 2000 g for 5 rain. The clear supernatant obtained after pH adjustment to 6.1-6.3 with 5 M K2CO 3 and centrifugation at 2000 g for 5 min, was passed through an Amberlite CG-50 column (0.2 ml). The column was washed twice with 1 ml of H20 and once with 0.25 ml of 0.5 M HCI. Amines were eluted with 0.5 ml of 0.5 M HCI and analyzed by high-performance liquid chromatography (Yanaco, ODS-T column) with a voltammetry detector (Yanaco, VMD-101 A). The mobile phases were 0.1 M potassium phosphate buffer for catecholamines and the buffer containing 10%. methanol for 5-HT. The flow rate was 0.8 ml/min and the detector potential was set at 0.8 V against the Ag/AgCI electrode. Amino acid neurotransmitters were assayed by the
Correspondence: T. Suzuki, Department of Biochemistry, Nagoya City University Medical School, Mizuho-Ku, Nagoya 467, Japan.
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
375 method of Dodd et al. 7. To the synaptosomal supernatant, 5 nmol of glucosaminic acid as internal standard and 0.2 N perchloric acid were added, and the protein precipitated was removed by centrifugation at 2000 g for 5 min. Amino acids in the supernatant were analyzed by amino acid analyzer. LDH was assayed by the method of Kornberg 11. Proteins were determined by the method of Lowry et al. 12with bovine serum albumin as standard. Synaptolysis was followed by the determination of LDH liberated from synaptosomes into the incubation medium during 6-OHDA treatment. L D H activity was higher in the medium of 6 - O H D A treatment than in that of the control (data not shown), suggesting that the treatment induced synaptolysis. The specificity of 6 - O H D A toxicity was examined by the assay of catecholamines, 5-HT, and amino acid neurotransmitters liberated from synaptosomes. Large amounts of catecholamines were liberated by 6-OHDA as shown in Fig. 1. The time courses of the catecholamine liberation were similar to that of L D H liberation (not shown), rising immediately from the 0-min incubation and reaching a plateau in 30 min. The amounts of dopamine and noradrenaline liberated after 30 min incubation were 72% and 25%, respectively, of the total amounts contained in synapto-
a
f
.a o
b
TABLE I
Amino acid contents in the cerebral synaptosomes The values were average of the triplicate experiments.
Amino acid
Contents (nmol/g wet wt. of cerebrum)
Tau* Asp* Thr* Ser* Glu* Pro Gly* Ala* Val Met Ile Leu Tyr Phe Lys His Arg GABA*
227 423 37.9 58.0 444 35.6 64.6 52.7 7.5 6.3 9.0 14.3 7.1 31.2 20.6 7.9 20.8 184
* Putative amino acid neurotransmitters.
somes. The amounts of 5-HT liberated by 6 - O H D A were the same as those of the controls (Fig. lb), and were less than 10% of the total in synaptosomes. The amino acid contents of synaptosomes are shown in Table I. Amino acid neurotransmitters, especially taurine, aspartic acid, glutamic acid, and G A B A , were abundant in synaptosomes. The liberation of these amino acids were examined in the presence or absence of 150/zM 6-OHDA (Fig. 2). AI-
100 (%) lOO
i
= 0
u
;2o ~g
.E
0
10
~'eTa u
70 .
20
30
tn~ 0
10
30 60
Glu
..A--'"
20
AI~
201
0
a,~ "~
F
Incubation time (min)
Fig. 1. Liberation of catecholamines and 5-HT from synaptosomes by 6-OHDA. The amounts of catecholamines and 5-HT liberated into the media were assayed in triplicate by high-performance liquid chromatograph with voltammetry. For the detailed procedures see the text. a: catecholamine liberation from synaptosomes by treatment with 150/zM 6 - O H D A at 30 °C. b: liberation of 5-HT by treatment at 30 °C. ~ , dopamine liberated by 6 - O H D A ; - - O - - , dopamine (control); --& , noradrenaline liberated by 6-OHDA; - - A - -, noradrenaline (control); • , 5-HT liberated by 6-OHDA; - - [] - -, 5-HT (control).
_=
s_
~__
GA.A
1 ~0 O.
10
20
30 Incubation
0
0
,
,
,
10
20
30
Time (min)
Fig. 2. Liberation of amino acid neurotransmitters from synaptosomes by 6 - O H D A . The liberation of amino acid neurotransmitters by 6 - O H D A (150¢tM) at 30 °C into the incubation media were assayed in triplicate by an amino acid analyzer. For the detailed procedures see the text. , 6-OHDAtreated; . . . . , control.
376 though the amino acid liberation increased during the
to the transmitter contained 5,~,. Large amounts of do-
incubation, the difference in contents between 6 - O H D A treatment and the control was insignificant. The amounts of amino acids liberated by 6O H D A after 30 rain were 2.4%, 1.4%, 1.0%, 3.2%, 0.3%, 2.8%, 2.4%, and 2.7% of the total contents in the case of taurine, glutamic acid, aspartic acid, threonine, G A B A , alanine, serine and glycine, re-
pamine and noradrenaline together with LDH were liberated from synaptosomes during incubation in the presence of the drug, whereas neither 5-HT nor amino acid neurotransmitters escaped from the synaptosomes. The results indicated that catecholaminergic synaptosomes were destroyed by 6OHDA.
spectively. The present work demonstrates 6 - O H D A toxicity
The present investigation was supported in part by
is specific to the catecholaminergic synaptosomes prepared from rat cerebra. The synaptosomes pre-
the Grant-in-Aid for Scientific Research from the Japanese Ministry of Education.
pared from the brain are heterogeneous with respect
1 Angeletti, P. U. and Levi-Montalcini, R. Sympathetic nerve cell destruction in newborn mammals by 6-hydroxydopamine, Proc. nat. Acad. Sci. U.S.A., 65 (1970) 114-121. 2 Angeletti, P. U. and Levi-Montalcini, R., Cytolytic effect of 6-hydroxydopamine on neuroblastoma cells, Cancer Res., 30 (1970) 2863-2869. 3 Burnstock, G., Morphological changes produced by drugs acting on the autonomic nervous system, Pharmacol. Ther., 5 (1979) 49-53. 4 Cotman, C. W. and Matthews, D. A., Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization, Biochim. biophys. Acta., 249 (1971) 380-394. 5 Docherty, M., Bradford, H. and Anderton, B., Lysis of cholinergic synaptosomes by an antiserum to choline acetyltransferase, FEBS Lett., 144 (1982) 47-50. 6 Docherty, M., Bradford, H. F., Anderton, B. and Wu, JY., Specificlysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase, FEBS Lett., 152 (1983) 57-61. 7 Dodd, P. R., Hardy, J. A., Oakley, A. E., Edwardson, J. A., Perry, E. K. and Delaunoy, J.-P., A rapid method for preparing synaptosomes: comparison with alternative procedures, Brain Research, 226 (1981) 107-118. 8 Fried, R. C. and Blaustein, M. P., Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes), J. Cell Biol., 78 (1978) 685-700. 9 Heikilla, R. and Cohen, G., Inhibition of biogenic amine uptake by hydrogen peroxide: a mechanism for toxic effects of 6-hydroxydopamine, Science, 172 (1971) 1257-1258. 10 Jonsson, G. and Sachs, C., Effects of 6-hydroxydopamine on the uptake and storage of noradrenaline in sympathetic adrenergic neurons, Europ. J. Pharmacol., 9 (1970)
141-155. 11 Kornberg, A., Methods in Enzymology. 1, Academic Press, New York, 1955, pp. 441-443. 12 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 13 Nagatsu, T., Oka, K. and Kato, T., Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography, J. Chromatogr., 163 (1979) 247-252. 14 Prasad, K. N., Effect of dopamine and 6-hydroxydopamine on mouse neuroblastoma cells in vitro, Cancer Res., 31 (1971) 1457-1460. 15 Sachs, C., Jonsson, G., Heikkila, R. and Cohen, G., Control of the neurotoxicity of 6-hydroxydopamine by intraneuronal noradrenaline in rat iris, Acta physiol, scand., 93 (1975) 345-351. 16 Thoenen, H. and Tranzer, J. P., Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine, Naunyn-Schmiedebergs Arch. Pharmacol., 261 (1968) 271-288. 17 Tiffany-Castiglioni, E. and Perez-Polo, J. R., Evaluation of methods for determining 6-hydroxydopamine cytotoxicity, In Vitro, 16 (1980) 59t-599. 18 Tiffany-Castiglioni, E., Saneto, R. P., Proctor, P. H. and Perez-Polo, J. R., Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line, Biochem. Pharmacol., 31 (1982) 181-188. 19 Tiffany-Castiglioni, E. and Perez-Polo, J. R., The role of nerve growth factor in vitro in cell resistence to 6-hydroxydopamine toxicity, Exp. CellRes., 121 (1979) 179-189. 20 Tranzer, J. P. and Thoenen, H., An electron microscopic study of selective, acute degeneration of sympathetic nerve terminals after administration of 6-hydroxydopamine. Experientia, 24 (1968) 155-156.