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on apomorphine-induced stereotyped cage-climbing and on striatal dopamine release in mice. Comparative studies with amphetamine
Neuropeptides 3: 91-96,1982
EFFECTS OF b-/TYRg/MELANOTROPIN-/g-18/ ON APOMORPHINE-INDUCED STEREOTYPED CAGE-CLIMBING AND ON STRIATAL DOPAMINE RELEASE IN MICE. COMPARATIVE STUDIES WITH AMPHETAMINE L&1el6 Vecsei, Gyula Telegdy, Andrew V. SchallyX and David H. Coyx De artment of Pathophysiology, University Medical School, H- %701 Szeged, P.O.B. 531, Hungary XEndocrine and Polypeptide Laboratory, Veterans Administration Medical Center and Tulane University School of Medicine, New Orleans, Louisiana, U.S.A. /reprint request to GT/ ABSTRACT h-/Tyr'/melanotropin-/g-18/ significantly increased apomorphine-induced cage-climbing, while only a tendency towards an increase was observed after supersensitization with haloperidol. Amphetamine also enhanced apomorphine-induced climbing behavior. The peptide showed only a tendency to increase dopamine release from striatal slices, while amphetamine elevated this significantly. The results suggest that the striatal dopamine system is involved in the behavioral action of &/Tyrg/ melanotropin-/9-18/o INTRODUCTION &/Tyr'/melanotropin-/g-18/ was discovered in pig hypothalamus by Schally et al. /l/. It has the following amino-acid sequence: H-Tyr-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp-OH. This decapeptide also has a tetrapeptide sequence of amino-acids /Phe-Arg-Trp-Gly/ common to the 7-10 sequence in corticotropin /ACTH/ and MSH. Plotnikoff and Kastin /2/ reported that the structurally related MSH had the ability to potentiate the behavioral effects of DOPA. Lichtensteiger and Monnet /3/ have found a correlation between o(-MSH administration and the dopamine neurons of the substanoia nigra. A high dose ofO(-MSH caused a decrease in the cellular fluorescence intensity of the nigral dopamine neurons in males. In our earlier investigations on rats it was shown that &/Tyrg/melanotropin-/g-18/ increased the passive avoidance latency after pretrial treatment, inhibited the extinction 91
of active avoidance behavior and increased the open-field activity /4/. Haloperidol, a relatively specific dopamine receptor blocking agent /5/, completely blocked the open-field activation induced by the peptide /6/. In the neurochemical experiments a lower dopamine concentration was found in the septum after &-methyl-p-tyrosine administration and there were opposite effects on the dopamine concentration of the striatum /7/. These results suggest that the dopaminergic system might be involved in the behavioral action of /S-/Tyrg/melanotropin-/g-10/. Protais et al. /8/ demonstrated that the cage-climbing steretyped behavior may represent a convenient means of assessing the stimulation of striatal dopamine receptors in mice. Thus, the prgsent study was intended to investigate the effect of &/Tyr /melanotropin-/g-18/ on cage-climbing behavior in order to acquire information about its influence on the nigro-striatal dopaminergic system. Furthermore, Sandman an Kastin /9/ reported that, although d-MSH and d-amphetamine influence different behaviors, they may interact to potentiate some behaviors* In the open-field test A-MSH and d-amphetamine may affect similar sites in the brain. It is also known that amphetamine induces release of endogenous dopamine from corpus striatum /lo/. These data induced s to perform experiments to compare the actions of 4-/Tyr Y/melanotropin-/g-18/ and amphetamine on cage-climbing behavior and on the release of dopamine from striatal slices. MATERIALS AND METHODS Peptide &/Tyr'/melanotropin-/g-18/ was synthetized by solid-phase methods as previously described /l/. Experimental animals Adult male CFLP mice of an inbred strain were used. The weight of the animals was 25-35 g and they were housed 5 per cage. They were kept under a standard 12 h illumination schedule /light on at 6.00 am/. Food and water were available ad libitum. Behavioral method Climbing behavior was described by Protais et al. /8/ as a simple test for studying dopamine-receptor activity in the striatum. Briefly, the mice were treated with apomorphine hydrochloride /l.O or 0.5 mg/kg SC./, and were then placed individually in wire-meshed cages. After a habituation period of 5 min, caga-climbing behavior was observed for the next 10 min /8/. The peptide /lOO pg/mouse ip./ or d,l-amphetamine /l.O or 4.0 mg/kg ip./ was administered 30 min before apomorphine /0.5 or 1.0 mg/kg SC./ administration. Haloperidol was injected /4.0 mg/kg ip,/ 72 h before the session. 92
Biochemical method Dopamine release was investigated as described in detail by Darden and Hunt /ll/. Normal untreated mice were decapitated and the striatum was dissected. The tissue was chopped on ice into l-2 mg slices. The slices were preincubated for 10 min in KRBG buffer not onta'ning EDTA. The slices were then incubated with 5 x 10-9 M /gH/-Dopamine, /Amersham, England specific activity: 6.3 Ci/mmol/ for 10 min. After being washed'and centrifuged, the slices were reincubated for 20 min in order to remove the radioactivity no8 taken up actively. Finally, the slices were incubated at 37 C for 3 min in KRBG buffer containing 4.2 mM potassium and /S-/Tyrg /melanotropin-/g-18/ or amphetamine. After centrifugation, the medium /supernatant/ and the pellet were separated. The pellets were homogenized in absolute ethanol. Radioactivity was measured separately in the medium and in the pellet fraction. The release of /3H/DA was expressed as the ratio dpm medium/dpm tissue. Drugs d,l-Amphetamine-phosphate /CHINOIN, Budapest/, Haloperidol /Gedeon RICHTER, Budapest/ and Apomorphine-hydrochloride were purchased from commercial sources. All drgus were freshly dissolved in 0.9% saline before use, with exception of apomorphine. This was dissolved in 0.1% ascorbic acid diluted with saline. Statistical analysis Biochemical data were analysed by means of Student's t-test. The U-test of Mann-Whitney was used to evaluate behavioral data. Results &/Tyrg/melanotropin-/g-18/ /lOO pg/mouse/ increased the apomorphine-induced cage-climbing behavior /p40,05/. However, only a tendency to increase could be observed when the combined treatment of the peptide and apomorphine was applied after supersensitization by haloperidol. Amphetamine in a dose of 4.0 mg/kg significantly increased the stereotyped behavior /p
Table I. Effects of &/Tyr'/melanotropin-/g-18/ and amphetamine on apomorphine-induced stereotyped cage-climbing behavior Treatment
score/min
Apomor hine /l.O mg/kg/ P-/Tyr g/MSHg-18/100 pg/mouse/ + Apomorphine /l.O mg/kg/ Haloperidol /4.0 mg/kg/ + Apomorphine /0.5 my/kg/ Haloperidol /4.0 mg/kg/ + b-/Tyr /MSH _ 8 /lOO ug/mouse/ + Apomorphine /0.5 mg/ Ug Apomorphine /l.O mg/kg/ Amphetamine /l.O mg/kg/ + Apomorphine LO m&kg/ Amphetamine /4.0 mg/kg/ + Apomorphine /L.O mg/kg/
8.1~1.5 /lo/ 18.721.9 /lO/x 8.5kl.9 /lo/ 12.121.6 /ll/ 9.Q1.2
/18/
10.221.3 /ll/ 17.5~1.8 /lO/x
/ / Number of animals used‘ Asterisks represent significant difference /x = p&0,05/
Table II. Effects of amphetamine and f3-/Ty '/melanotropin-/918/ on the in vitro release of /TH/dopamine by striatal slices concentration /M/ control lO-6 M lO-5 M lO-4 M
Amphetamine DPM medium /4&~y;$~~g-18 2.720.4 /15/ 5.3kO.4 /8/x 8.3~0.7 /8/= 17.6kO.9 /7/-
2.9LO.5 /14/ 3.1~0.4 /8/ 3.320.4 /9/ 3.6kO.3 /9/
/ / Number of animals used. Asterisks represent significant difference /x = ~40.05; xx = ~(0.01; xxx = p!tO,OOl/. which indicates a dopamine-mediated effect of MSH. &-/Tyrg/melanotropin-/g-18/, which has a common amino-acid sequence with ACTH/MSH7_10 /l/, increased the apomorphine-induced cage-climbing behavior. This decapeptide also increased the dopamine concentration of the striatum after oGMPT pretreatment ese behavioral and neurochemical data indicate that /7/. Tjj p-/Tyr /melanotropin-/g-18/ acts on the nigro-striatal system; however, the mechanism of action remains obscure. Amphetamine-induced potentiation of the climbing behavior was reported by Martes et al. /13/. In accordance with this
94
result, with a high dose of amphetamine /4 x 5 mg/kg/ it potentiated the climbing activity of mice. Further, in our experiments both amphetamine and the peptide potentiated the apomorphine-induced climbing behavior of the animals. The effect of amp etamine in this experiment was similar to that of $ /melanotropin-/g-18/ just like in the open-field test I;--$Tyr ;Iowever, amphetamine strongly increased the release of dopamine from striatal slices of mice, whereas the peptide showed only a tendency to increase it. The data suggest that while amphetamine incr ases the cage-climbing activity by releasing dopamine, (J-/Tyr%/melanotropin-/g-18/ presumably influences it by increasing the sensitivity of the receptor sites. After haloperidol supersensitization, only a tendency to a further increase could be achieved by subsequent treatment with the peptide. This result supported the earlier conclusion. REFERENCES 1.
Schally,A.V., Chang,R.C.C., Huang,W.Y., Coy,D.H., Kastin,A. biologJ. and Redding,T.W. /1980/. Isolation, struct ical characterizatin, and synthesis of &/Tyr ye, /melanotropin-/g-18/ decapeptide from pig hypothalami. Proc.natn.Acad. Sci.USA 77: 3947-3951.
2. Plotnikoff,N.P. and Kastin,A.J. /1976/ Neuropharmacological tests with o(-melanocyte stimulating hormone. Life Sci. 18: 1217-1222.
3. Lichtensteiger,W. and Monnet,F. /1979/ Differential response of dopamine neurons to ti-melanotropin and analogues in relation to their endocrine and behavioral potency. Life Sci. 25: 2079-2087.
4. VQcsei,L., Telegd g,G., Schally,A.V. and Coy,D.H. /1981/. Effects of &/Tyr /melanotropin-/g-18/ decapeptide on passive and active avoidance behavior and on open-field activity of rats. Peptides 2: 389-391.
5. AndBn,N.E., Butcher,S.G., Corrodi,H., Fuxe,K, and Ungerstedt,U. /1970/. Receptor activity and turnover of dopamine and noradrenaline after neuroleptics. European J.Pharmacol.
11: 303-314. 6. Telegdy,G., VQcsei,L., Schally A.V. and Coy,D.H. /1981/. The interaction between &/Tyr9/melanotropin-/g-18/, haloperidol and amphetamine in different behavior tests of rats. Pharmac.Biochem.Behav. 17: 15-18.
7. Fekete,M., Telegd ,G., Schally,A.V. and Coy,D.H. /1981/.
Effects of /&-/Tyr3/melanotropin-/g-18/ decapeptide on catecholamine disappearance and serotonin accumulation in discrete brain regions of rats. Neuropeptides 1: 377-382.
95
8. Protais,P., Costentin,J. and Schwartz,J.C. /1976/. Climbing behavior induced by apomorphine in mice: a simple test for the study of dopamine receptors in striatum. Psychopharmacology 50: 1-6. 9. Sandman,C.A. and Kastin,A.J. /1978/. Interaction of o(-MSH and MIF-I with d-amphetamine on open-field behavior of rats. Pharmac.Biochem.Behav. 9: 759-762. 10. Meyerhoff,J.L. and Kant,G.J. /1978/. Release of endogenous dopamine from corpus striatum. Life. Sci. 23: 1481-1486. 11. Darden,J.H. and Hunt,W.A. /1977/. Reduction of striatal dopamine release during ethanol withdrawal syndrome. J. Neurochem. 29: 1143-1145. 12. De Wied,D. and Gispen,W.H. /1977/. Behavioral effects of peptides. In: Gainer,H. /ed ./ Peptides in Neurobiology, Plenum Press, New York, p. 397-448. 13. Martes,M.P., Costentin,J., Baudry,J., Marcais,H., Protais, P. and Schwartz,J.C. /1977/. Long-term changes in the sensitivity of pre- and postsynaptic dopamine receptors in mouse striatum evidenced by behavioural and biochemical studies. Brain Res. 136: 319-327.
Received Accepted
20 October lg82 25 October 1982
96
EFFECTS OF b-/TYRg/MELANOTROPIN-/g-18/ ON APOMORPHINE-INDUCED STEREOTYPED CAGE-CLIMBING AND ON STRIATAL DOPAMINE RELEASE IN MICE. COMPARATIVE STUDIES WITH AMPHETAMINE L&1el6 Vecsei, Gyula Telegdy, Andrew V. SchallyX and David H. Coyx De artment of Pathophysiology, University Medical School, H- %701 Szeged, P.O.B. 531, Hungary XEndocrine and Polypeptide Laboratory, Veterans Administration Medical Center and Tulane University School of Medicine, New Orleans, Louisiana, U.S.A. /reprint request to GT/ ABSTRACT h-/Tyr'/melanotropin-/g-18/ significantly increased apomorphine-induced cage-climbing, while only a tendency towards an increase was observed after supersensitization with haloperidol. Amphetamine also enhanced apomorphine-induced climbing behavior. The peptide showed only a tendency to increase dopamine release from striatal slices, while amphetamine elevated this significantly. The results suggest that the striatal dopamine system is involved in the behavioral action of &/Tyrg/ melanotropin-/9-18/o INTRODUCTION &/Tyr'/melanotropin-/g-18/ was discovered in pig hypothalamus by Schally et al. /l/. It has the following amino-acid sequence: H-Tyr-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp-OH. This decapeptide also has a tetrapeptide sequence of amino-acids /Phe-Arg-Trp-Gly/ common to the 7-10 sequence in corticotropin /ACTH/ and MSH. Plotnikoff and Kastin /2/ reported that the structurally related MSH had the ability to potentiate the behavioral effects of DOPA. Lichtensteiger and Monnet /3/ have found a correlation between o(-MSH administration and the dopamine neurons of the substanoia nigra. A high dose ofO(-MSH caused a decrease in the cellular fluorescence intensity of the nigral dopamine neurons in males. In our earlier investigations on rats it was shown that &/Tyrg/melanotropin-/g-18/ increased the passive avoidance latency after pretrial treatment, inhibited the extinction 91
of active avoidance behavior and increased the open-field activity /4/. Haloperidol, a relatively specific dopamine receptor blocking agent /5/, completely blocked the open-field activation induced by the peptide /6/. In the neurochemical experiments a lower dopamine concentration was found in the septum after &-methyl-p-tyrosine administration and there were opposite effects on the dopamine concentration of the striatum /7/. These results suggest that the dopaminergic system might be involved in the behavioral action of /S-/Tyrg/melanotropin-/g-10/. Protais et al. /8/ demonstrated that the cage-climbing steretyped behavior may represent a convenient means of assessing the stimulation of striatal dopamine receptors in mice. Thus, the prgsent study was intended to investigate the effect of &/Tyr /melanotropin-/g-18/ on cage-climbing behavior in order to acquire information about its influence on the nigro-striatal dopaminergic system. Furthermore, Sandman an Kastin /9/ reported that, although d-MSH and d-amphetamine influence different behaviors, they may interact to potentiate some behaviors* In the open-field test A-MSH and d-amphetamine may affect similar sites in the brain. It is also known that amphetamine induces release of endogenous dopamine from corpus striatum /lo/. These data induced s to perform experiments to compare the actions of 4-/Tyr Y/melanotropin-/g-18/ and amphetamine on cage-climbing behavior and on the release of dopamine from striatal slices. MATERIALS AND METHODS Peptide &/Tyr'/melanotropin-/g-18/ was synthetized by solid-phase methods as previously described /l/. Experimental animals Adult male CFLP mice of an inbred strain were used. The weight of the animals was 25-35 g and they were housed 5 per cage. They were kept under a standard 12 h illumination schedule /light on at 6.00 am/. Food and water were available ad libitum. Behavioral method Climbing behavior was described by Protais et al. /8/ as a simple test for studying dopamine-receptor activity in the striatum. Briefly, the mice were treated with apomorphine hydrochloride /l.O or 0.5 mg/kg SC./, and were then placed individually in wire-meshed cages. After a habituation period of 5 min, caga-climbing behavior was observed for the next 10 min /8/. The peptide /lOO pg/mouse ip./ or d,l-amphetamine /l.O or 4.0 mg/kg ip./ was administered 30 min before apomorphine /0.5 or 1.0 mg/kg SC./ administration. Haloperidol was injected /4.0 mg/kg ip,/ 72 h before the session. 92
Biochemical method Dopamine release was investigated as described in detail by Darden and Hunt /ll/. Normal untreated mice were decapitated and the striatum was dissected. The tissue was chopped on ice into l-2 mg slices. The slices were preincubated for 10 min in KRBG buffer not onta'ning EDTA. The slices were then incubated with 5 x 10-9 M /gH/-Dopamine, /Amersham, England specific activity: 6.3 Ci/mmol/ for 10 min. After being washed'and centrifuged, the slices were reincubated for 20 min in order to remove the radioactivity no8 taken up actively. Finally, the slices were incubated at 37 C for 3 min in KRBG buffer containing 4.2 mM potassium and /S-/Tyrg /melanotropin-/g-18/ or amphetamine. After centrifugation, the medium /supernatant/ and the pellet were separated. The pellets were homogenized in absolute ethanol. Radioactivity was measured separately in the medium and in the pellet fraction. The release of /3H/DA was expressed as the ratio dpm medium/dpm tissue. Drugs d,l-Amphetamine-phosphate /CHINOIN, Budapest/, Haloperidol /Gedeon RICHTER, Budapest/ and Apomorphine-hydrochloride were purchased from commercial sources. All drgus were freshly dissolved in 0.9% saline before use, with exception of apomorphine. This was dissolved in 0.1% ascorbic acid diluted with saline. Statistical analysis Biochemical data were analysed by means of Student's t-test. The U-test of Mann-Whitney was used to evaluate behavioral data. Results &/Tyrg/melanotropin-/g-18/ /lOO pg/mouse/ increased the apomorphine-induced cage-climbing behavior /p40,05/. However, only a tendency to increase could be observed when the combined treatment of the peptide and apomorphine was applied after supersensitization by haloperidol. Amphetamine in a dose of 4.0 mg/kg significantly increased the stereotyped behavior /p
Table I. Effects of &/Tyr'/melanotropin-/g-18/ and amphetamine on apomorphine-induced stereotyped cage-climbing behavior Treatment
score/min
Apomor hine /l.O mg/kg/ P-/Tyr g/MSHg-18/100 pg/mouse/ + Apomorphine /l.O mg/kg/ Haloperidol /4.0 mg/kg/ + Apomorphine /0.5 my/kg/ Haloperidol /4.0 mg/kg/ + b-/Tyr /MSH _ 8 /lOO ug/mouse/ + Apomorphine /0.5 mg/ Ug Apomorphine /l.O mg/kg/ Amphetamine /l.O mg/kg/ + Apomorphine LO m&kg/ Amphetamine /4.0 mg/kg/ + Apomorphine /L.O mg/kg/
8.1~1.5 /lo/ 18.721.9 /lO/x 8.5kl.9 /lo/ 12.121.6 /ll/ 9.Q1.2
/18/
10.221.3 /ll/ 17.5~1.8 /lO/x
/ / Number of animals used‘ Asterisks represent significant difference /x = p&0,05/
Table II. Effects of amphetamine and f3-/Ty '/melanotropin-/918/ on the in vitro release of /TH/dopamine by striatal slices concentration /M/ control lO-6 M lO-5 M lO-4 M
Amphetamine DPM medium /4&~y;$~~g-18 2.720.4 /15/ 5.3kO.4 /8/x 8.3~0.7 /8/= 17.6kO.9 /7/-
2.9LO.5 /14/ 3.1~0.4 /8/ 3.320.4 /9/ 3.6kO.3 /9/
/ / Number of animals used. Asterisks represent significant difference /x = ~40.05; xx = ~(0.01; xxx = p!tO,OOl/. which indicates a dopamine-mediated effect of MSH. &-/Tyrg/melanotropin-/g-18/, which has a common amino-acid sequence with ACTH/MSH7_10 /l/, increased the apomorphine-induced cage-climbing behavior. This decapeptide also increased the dopamine concentration of the striatum after oGMPT pretreatment ese behavioral and neurochemical data indicate that /7/. Tjj p-/Tyr /melanotropin-/g-18/ acts on the nigro-striatal system; however, the mechanism of action remains obscure. Amphetamine-induced potentiation of the climbing behavior was reported by Martes et al. /13/. In accordance with this
94
result, with a high dose of amphetamine /4 x 5 mg/kg/ it potentiated the climbing activity of mice. Further, in our experiments both amphetamine and the peptide potentiated the apomorphine-induced climbing behavior of the animals. The effect of amp etamine in this experiment was similar to that of $ /melanotropin-/g-18/ just like in the open-field test I;--$Tyr ;Iowever, amphetamine strongly increased the release of dopamine from striatal slices of mice, whereas the peptide showed only a tendency to increase it. The data suggest that while amphetamine incr ases the cage-climbing activity by releasing dopamine, (J-/Tyr%/melanotropin-/g-18/ presumably influences it by increasing the sensitivity of the receptor sites. After haloperidol supersensitization, only a tendency to a further increase could be achieved by subsequent treatment with the peptide. This result supported the earlier conclusion. REFERENCES 1.
Schally,A.V., Chang,R.C.C., Huang,W.Y., Coy,D.H., Kastin,A. biologJ. and Redding,T.W. /1980/. Isolation, struct ical characterizatin, and synthesis of &/Tyr ye, /melanotropin-/g-18/ decapeptide from pig hypothalami. Proc.natn.Acad. Sci.USA 77: 3947-3951.
2. Plotnikoff,N.P. and Kastin,A.J. /1976/ Neuropharmacological tests with o(-melanocyte stimulating hormone. Life Sci. 18: 1217-1222.
3. Lichtensteiger,W. and Monnet,F. /1979/ Differential response of dopamine neurons to ti-melanotropin and analogues in relation to their endocrine and behavioral potency. Life Sci. 25: 2079-2087.
4. VQcsei,L., Telegd g,G., Schally,A.V. and Coy,D.H. /1981/. Effects of &/Tyr /melanotropin-/g-18/ decapeptide on passive and active avoidance behavior and on open-field activity of rats. Peptides 2: 389-391.
5. AndBn,N.E., Butcher,S.G., Corrodi,H., Fuxe,K, and Ungerstedt,U. /1970/. Receptor activity and turnover of dopamine and noradrenaline after neuroleptics. European J.Pharmacol.
11: 303-314. 6. Telegdy,G., VQcsei,L., Schally A.V. and Coy,D.H. /1981/. The interaction between &/Tyr9/melanotropin-/g-18/, haloperidol and amphetamine in different behavior tests of rats. Pharmac.Biochem.Behav. 17: 15-18.
7. Fekete,M., Telegd ,G., Schally,A.V. and Coy,D.H. /1981/.
Effects of /&-/Tyr3/melanotropin-/g-18/ decapeptide on catecholamine disappearance and serotonin accumulation in discrete brain regions of rats. Neuropeptides 1: 377-382.
95
8. Protais,P., Costentin,J. and Schwartz,J.C. /1976/. Climbing behavior induced by apomorphine in mice: a simple test for the study of dopamine receptors in striatum. Psychopharmacology 50: 1-6. 9. Sandman,C.A. and Kastin,A.J. /1978/. Interaction of o(-MSH and MIF-I with d-amphetamine on open-field behavior of rats. Pharmac.Biochem.Behav. 9: 759-762. 10. Meyerhoff,J.L. and Kant,G.J. /1978/. Release of endogenous dopamine from corpus striatum. Life. Sci. 23: 1481-1486. 11. Darden,J.H. and Hunt,W.A. /1977/. Reduction of striatal dopamine release during ethanol withdrawal syndrome. J. Neurochem. 29: 1143-1145. 12. De Wied,D. and Gispen,W.H. /1977/. Behavioral effects of peptides. In: Gainer,H. /ed ./ Peptides in Neurobiology, Plenum Press, New York, p. 397-448. 13. Martes,M.P., Costentin,J., Baudry,J., Marcais,H., Protais, P. and Schwartz,J.C. /1977/. Long-term changes in the sensitivity of pre- and postsynaptic dopamine receptors in mouse striatum evidenced by behavioural and biochemical studies. Brain Res. 136: 319-327.
Received Accepted
20 October lg82 25 October 1982
96