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Effects of agents increasing serotonin transmission on the increase of dopamine metabolism caused by morphine in the rat nucleus accumbens
Pharmacolegical Research Communications, Vol. 16, No. 5, 1984
51 3
EFFECTS OF AGENTS INCREASING SEROTONIN TRANSMISSION ON THE INCREASE OF DOPAMINE METABOLISM CAUSED BY MORPHINE IN THE RAT NUCLEUS ACCUMBENS
U. Spampinato,
E. Nowakowska
, R. Samanin
Istituto di Ricerche Farmacologiche Via Eritrea 62,
"Mario Negri"
20157 MILAN, Italy
Received~final ~ r m 22 December 1983 SUMMARY
The effect of subcutaneous injection of IO mg/kg morphine on homovanillic acid (HVA) concentrations in the nucleus accumbens was studied in rats which had received d-fenfluramine, a serotonin releaser, or m-chlorophenylpiperazine, a serotonin agonist, before morphine. 2.5 mg/kg d-fenfluramine or m-chlorophenylpiperazine had no effect on HVA in the nucleus accumbens but significantly reduced the rise in HVA induced by morphine. None of the drugs modified the levels of morphine in the rat brain. The results suggest that agents increasing serotonin transmission inhibit the effect of morphine on dopamine neurons innervating the nucleus accumbens. INTRODUCTION Serotonin
(5-HT) antagonists were recently reported to block morphine-
induced inhibition of hyperactivity nucleus
accumbens
injections
caused by dopamine
(Costall et al., 1977).
in the nucleus accumbens
DA (Costall et al., 1976),
(DA) injected in the
Together with the fact that 5-HT
inhibit the effect of locally applied
these findings suggest that morphine-induced
activation of 5-HT mechanisms
(Miranda et al., 1979; Snelgar and Vogt,
1981)
opposes dopamine activity on postsynaptic neuronal elements in the nucleus accumbens. A single injection of morphine increases
the metabolism of DA in
the nucleus accumbens by a mechanism which is not completely clear (Westerink and
Korf, 1976).
Since 5-HT injection in this area raises
the levels of homovanillic acid, a major DA metabolite
(Pycock et al.,
1978), possibly part of morp]~ine's effect on DA metabolism
i Visiting Scientist from Dept. of Pharmacology of Sciences, Poznam, Poland • ~ To whom correspondence
is mediated
and Pharm.Medical
Academy
should be addressed
0031-6989/84/050513-05/$03.00/0
© 1984 The Italian Pharmacological Society
514
Pharmacological Research Communications, VoL 16, No. 5, 1984
by an action on 5-HT containing neurons.
The fact that 5-HT antagonists
and parachlorophenylalanine, an inhibitor of 5-HT synthesis (Koe and Weissman, 1966), partially prevent the morphine-induced increase of DA metabolism in the nucleus accumbens supports this hypothesis (Spampinato et al., 1984). The present study further examines the role of 5-HT
as
reflected
in the effect of morphine in animals treated with d-fenfluramine, a releaser of 5-HT from nerve terminals (Garattini et al., 1 9 7 9 ) a n d m-chlorophenylpiperazine (CPP), a potent 5-HT agonist (Samanin et al., 1979).
MATERIALS AND METHODS Male CD-COBS (Charles River, Italy) rats, weighing 175-200 g, were used.
They were kept at constant room temperature (21+I°C) and relative
humidity (60%) with a 12 hour light-12 hour dark cycle (dark period commencing at 7 p.m.).
The animals were injected subcutaneously with i0
mg/kg morphine hydrochloride (Farmitalia-Carlo Erba, Milan, Italy) and killed by decapitation i h later for biochemical assay,
2.5 mg/kg d-fenflu-
ramine hydrochloride (Servier Laboratory, Neuilly-sur-Seine, France) and 2.5 mg/kg CPP hydrochloride (Aldrich, Europe) were dissolved in saline and administered intraperitoneally 30 minutes before morphine.
The doses of
d-fenfluramine and CPP were selected on the basis of previous studies showing their significant effect on central serotonin mechanisms (Garattini et al., 1979; Invernizzi et al., 1981).
Homovanillic acid (HVA) was
measured in the nucleus accumbens by high performance liquid chromatography with electrochemical detection according to the method of Wightman et al. (1977) with minor modifications (Invernfzzi and Samanin, 1981).
Morphine
in the brain was measured according to a slighly modified version of the method described by Todd et al. (1982).
The data were statistically
analyzed by ANOVA 2 x 2 (Rocchetti and Recchia, 198~. F test for significant interaction were followed by Tukey's test to compare the experimental groups with controls.
RESULTS As shown in Table i, neither d-fenfluramine nor CPP significantly modified HVA levels in the nucleus accumbens.
Both drugs markedly reduced
the increase in HVA levels caused by morphine (F interaction p ~ 0.05).
In
order to check whether drugs had limited the effect of morphine by reducing
Pharmacological Research Communications, Vol. 16. No. 5, 1984
51 5
its entry into the brain, brain levels of morphine were measured in some animals,
taken randomly from the various group.
levels (measured I h after morphine
injection) was found in the brain of
treated animals compared with controls. ± S.E.
(mean of 6 determinations)
No decrease in morphine
Morphine concentrations
in ng/g
were: controls 281 + 9; d-fenfluramine
331 + 4; CPP 330 + 17.
Table i - Effects of d-fenfluramine (dF) and m-chlorophenylpiperazine (CPP) on the rise in homovanillic acid (HVA) levels induced by iO mg/kg morphine s.c. in the nucleus accumbens of rats. Treatment
(mg/kg)
HVA (ng/g + S.E.)
Saline + saline
367 + 14
d-F (2.5) + saline
440 + 16
saline + morphine
764 + 3 7 *
(iO)
d-F (2.5) + morphine
(i0)
6 4 5 + 28 °
saline + saline
3 7 2 + 19
CPP (2.5) + saline
3 4 6 + 35
saline + morphine
851 + 41"
(IO)
CPP (2.5) + morphine
639 + 30 °
(I0)
Eachvalue is the mean of 6 determinations. dF and CPP were administered intraperitoneally 30 minutes before morphine. The animals were killed I h after morphine injection. P KO.OI compared with saline + saline Op 40.05 (F interaction).
DISCUSSION d-Fenfluramine
and CPP significantly
on DA metabolism in the nucleus accumbens.
reduced the effect of morphine These findings were somewhat
surprising since the same effect was found in animals which had received 5-HT antagonists before morphine
(Spampinato et al., 1984).
One explanation
is that 5-HT antagonists mimic the action of 5-HT in the nucleus accumbens, an effect found with various 5-HT antagonists telencephalon
(Haigler and Aghajanian,
since parachlorophenylalanine, Weissman,
This, however,
an inhibitor of 5-HT synthesis
1966) also reduced morphine's
nucleus accumbens
1977).
in some regions of rat is unlikely, (Koe and
effect on DA metabolism
in the
(Spampinato et al., 1984) and there is evidence that
5-HT antagonists block the inhibitory action of 5-HT on dopamine-dependent
Pharmacological Research Communications, VoL 16, No. 5, 1984
516
effect Jn this area (Costall and Naylor, 1978). It has been shown recently that agents increasing 5-HT transmission reduce the morphine-induced increase of dopamine metabolism in the striatum of rats ($pampinato et al., 1983a).
This and other findings (Samanin et
al., 19~,~; Cervo et al., 1981; Spampinato et al., 1983b) have led to the suggestion that a single, moderate dose (I0 mg/kg) of morphine activates dopamine°-containing neurons in the striatum and that 5-HT mechanisms act presynaptically to inhibit this.
Morphine injected in the ventral teg-
mental ~rea (VTA) of rats has been reported to activate dopamine-containing neurons ~[nnervating the nucleus accumbens and other telencephalic regions (Joyce and Iversen, 1979) and 5-HT may influence dopamine mechanisms in the VTA (Beart and McDonald, 1982).
It is possible therefore that part of
morphine's effect on DA metabolism in the nucleus accumbens depends on its ability t~ activate DA-containing neurons in the VTA, and 5-HT mechanisms activated by d-fenfluramine and CPP act by inhibiting this effect.
The
apparent p~xadox of 5-HT antagonists and agonists having the same effect is therefore solved considering that morphine-induced activation of 5-HT mechanisms on postsynaptic elements in the nucleus accumbens may contribute to the increase in HVA (Costall and Naylor, 1978; Pycock et al., 1978) whereas 5-HT mechanisms controlling presynaptic DA activity reduce morphine's ability to activate dopamine-containing neurons in the rat brain (Samanin et al., 1980; Cervo et al., 1981; Spampinato et al., 1983b). That agents increasing 5-HT transmission inhibit morphine's effect on mesolimbic dopamine-containing neurons is of particular interest in view of the recent finding that the ventral tegmental area and the nucleus accumbens are involved in the reinforcing effects of opiates in rats (Olds, 1982; Britt and Wise, 1983).
ACKNOWLEDGEMENTS
This work was partially supported by Centro Nazionale delle Ricerche in the
contextof the Progetto Finalizzato "Medicina Proventiva e Riabili-
tativa": Sottoprogetto SP 7 "Tossicodipendenza". The authors are greatly indebted to Giancarlo Bianchi and Marco Landi for measuring morphine in the brain of the various experimental groups.
Pharmacological Research Communications, Vol. 16, No. 5, 1984 REFERENCES Beart P.M. and McDonald D. (1982) J. Pharm.Pharmac. 34: 591-593. Britt M.D. and Wise R.A. (1983) Brain Res. 258: 105-108. Cervo L., Romandini S. and Samanin R. (1981) Life Sci. 29: 2585-2591. Costall B., Fortune D.H. and Naylor R.J. (1977). Br. J. Pharmac. 60:266P. Costall B. and Naylor R.J. (1978) J. Pharm.Pharmac. 30: 257-259. Costall B., Naylor R.J., Marsden C.D. and Pycock C.J. (1976) J. Pharm. Pharmac. 28: 523-526. Garattini S., Caccia S., Mennini T., Samanin R., Consolo S. and Ladinsky H. (1979) Curr. med. Res. Opin. 6, suppl.l: 15-27. Haigler H.J. and Aghajanian G.K. (1977) Fed.Proc. 36: 2159-2164. Invernizzi R~, Cotecchia S., De Blasi A., Mennini T., Patacclni R. and Samanin R. (1981) Neurochem. Int. 3: 239-244. Invernizzi R. and Samanin R. (1981) Pharmac. Res. Commun. 13: 511-516. Joyce E.M.and Iversen S.D. (1979) Neurosei. Letters 14: 207-212. Koe B.K. and Weissman A. (1966) J. Pharmac. exp.Ther. 154: 449-517. Miranda F., Invernizzi R. and Samanin R. (1979) Pharmac. Res. Commun. ii: 455-466. Olds M.E. (1982) Brain Res. 237: 429-440. Pycock C.J., Horton R.W. and Carter C.J. (1978) Adv. Biochem.Psychopharmac. 19: 323-341. Rocchetti M. and Recchia M. (1982) Comput. Programs Biomed. 14: 7-20. Samanin R., Cervo L., Rochat C., Poggesi E. and Mennini T. (1980) Life Sci. 27: 1141-1146. Samanin R., Mennini T., Ferraris A., Bendotti C., Borsini F. and Garattini S. (1979) Naunyn Schmiedebergs' Arch. Pharmac. 308: 159-163. Snelgar R.S. and Vogt M. (1981) J. Physiol.(London) 314: 395-410. Spampinato U., Invernizzi R., Nowakowska E. and Samanin R. (1983a) Biochem. Pharmac. in press Spampinato U., Invernizzi R. and Samanin R. (1984) ~harmac. Res. Commun. This issue. SpamDinato U., Nowakowska E. and Samanin R. (1983b) J. Pharm. Pharmac. 35: 831-833. Todd R.D., Muldoon S.M. and Watson R.L. (1982) J. Chromatogr. 232: I01-ii0. Westerink B.H.C. and Korf J. (1976) Eur. J. Pharmac. 38: 281-291. Wightman R.M., Plotsky P.M., Strope E., Delcore R.Jr. and Adams R.N. (1977) Brain Res. 131: 345-349.
517
51 3
EFFECTS OF AGENTS INCREASING SEROTONIN TRANSMISSION ON THE INCREASE OF DOPAMINE METABOLISM CAUSED BY MORPHINE IN THE RAT NUCLEUS ACCUMBENS
U. Spampinato,
E. Nowakowska
, R. Samanin
Istituto di Ricerche Farmacologiche Via Eritrea 62,
"Mario Negri"
20157 MILAN, Italy
Received~final ~ r m 22 December 1983 SUMMARY
The effect of subcutaneous injection of IO mg/kg morphine on homovanillic acid (HVA) concentrations in the nucleus accumbens was studied in rats which had received d-fenfluramine, a serotonin releaser, or m-chlorophenylpiperazine, a serotonin agonist, before morphine. 2.5 mg/kg d-fenfluramine or m-chlorophenylpiperazine had no effect on HVA in the nucleus accumbens but significantly reduced the rise in HVA induced by morphine. None of the drugs modified the levels of morphine in the rat brain. The results suggest that agents increasing serotonin transmission inhibit the effect of morphine on dopamine neurons innervating the nucleus accumbens. INTRODUCTION Serotonin
(5-HT) antagonists were recently reported to block morphine-
induced inhibition of hyperactivity nucleus
accumbens
injections
caused by dopamine
(Costall et al., 1977).
in the nucleus accumbens
DA (Costall et al., 1976),
(DA) injected in the
Together with the fact that 5-HT
inhibit the effect of locally applied
these findings suggest that morphine-induced
activation of 5-HT mechanisms
(Miranda et al., 1979; Snelgar and Vogt,
1981)
opposes dopamine activity on postsynaptic neuronal elements in the nucleus accumbens. A single injection of morphine increases
the metabolism of DA in
the nucleus accumbens by a mechanism which is not completely clear (Westerink and
Korf, 1976).
Since 5-HT injection in this area raises
the levels of homovanillic acid, a major DA metabolite
(Pycock et al.,
1978), possibly part of morp]~ine's effect on DA metabolism
i Visiting Scientist from Dept. of Pharmacology of Sciences, Poznam, Poland • ~ To whom correspondence
is mediated
and Pharm.Medical
Academy
should be addressed
0031-6989/84/050513-05/$03.00/0
© 1984 The Italian Pharmacological Society
514
Pharmacological Research Communications, VoL 16, No. 5, 1984
by an action on 5-HT containing neurons.
The fact that 5-HT antagonists
and parachlorophenylalanine, an inhibitor of 5-HT synthesis (Koe and Weissman, 1966), partially prevent the morphine-induced increase of DA metabolism in the nucleus accumbens supports this hypothesis (Spampinato et al., 1984). The present study further examines the role of 5-HT
as
reflected
in the effect of morphine in animals treated with d-fenfluramine, a releaser of 5-HT from nerve terminals (Garattini et al., 1 9 7 9 ) a n d m-chlorophenylpiperazine (CPP), a potent 5-HT agonist (Samanin et al., 1979).
MATERIALS AND METHODS Male CD-COBS (Charles River, Italy) rats, weighing 175-200 g, were used.
They were kept at constant room temperature (21+I°C) and relative
humidity (60%) with a 12 hour light-12 hour dark cycle (dark period commencing at 7 p.m.).
The animals were injected subcutaneously with i0
mg/kg morphine hydrochloride (Farmitalia-Carlo Erba, Milan, Italy) and killed by decapitation i h later for biochemical assay,
2.5 mg/kg d-fenflu-
ramine hydrochloride (Servier Laboratory, Neuilly-sur-Seine, France) and 2.5 mg/kg CPP hydrochloride (Aldrich, Europe) were dissolved in saline and administered intraperitoneally 30 minutes before morphine.
The doses of
d-fenfluramine and CPP were selected on the basis of previous studies showing their significant effect on central serotonin mechanisms (Garattini et al., 1979; Invernizzi et al., 1981).
Homovanillic acid (HVA) was
measured in the nucleus accumbens by high performance liquid chromatography with electrochemical detection according to the method of Wightman et al. (1977) with minor modifications (Invernfzzi and Samanin, 1981).
Morphine
in the brain was measured according to a slighly modified version of the method described by Todd et al. (1982).
The data were statistically
analyzed by ANOVA 2 x 2 (Rocchetti and Recchia, 198~. F test for significant interaction were followed by Tukey's test to compare the experimental groups with controls.
RESULTS As shown in Table i, neither d-fenfluramine nor CPP significantly modified HVA levels in the nucleus accumbens.
Both drugs markedly reduced
the increase in HVA levels caused by morphine (F interaction p ~ 0.05).
In
order to check whether drugs had limited the effect of morphine by reducing
Pharmacological Research Communications, Vol. 16. No. 5, 1984
51 5
its entry into the brain, brain levels of morphine were measured in some animals,
taken randomly from the various group.
levels (measured I h after morphine
injection) was found in the brain of
treated animals compared with controls. ± S.E.
(mean of 6 determinations)
No decrease in morphine
Morphine concentrations
in ng/g
were: controls 281 + 9; d-fenfluramine
331 + 4; CPP 330 + 17.
Table i - Effects of d-fenfluramine (dF) and m-chlorophenylpiperazine (CPP) on the rise in homovanillic acid (HVA) levels induced by iO mg/kg morphine s.c. in the nucleus accumbens of rats. Treatment
(mg/kg)
HVA (ng/g + S.E.)
Saline + saline
367 + 14
d-F (2.5) + saline
440 + 16
saline + morphine
764 + 3 7 *
(iO)
d-F (2.5) + morphine
(i0)
6 4 5 + 28 °
saline + saline
3 7 2 + 19
CPP (2.5) + saline
3 4 6 + 35
saline + morphine
851 + 41"
(IO)
CPP (2.5) + morphine
639 + 30 °
(I0)
Eachvalue is the mean of 6 determinations. dF and CPP were administered intraperitoneally 30 minutes before morphine. The animals were killed I h after morphine injection. P KO.OI compared with saline + saline Op 40.05 (F interaction).
DISCUSSION d-Fenfluramine
and CPP significantly
on DA metabolism in the nucleus accumbens.
reduced the effect of morphine These findings were somewhat
surprising since the same effect was found in animals which had received 5-HT antagonists before morphine
(Spampinato et al., 1984).
One explanation
is that 5-HT antagonists mimic the action of 5-HT in the nucleus accumbens, an effect found with various 5-HT antagonists telencephalon
(Haigler and Aghajanian,
since parachlorophenylalanine, Weissman,
This, however,
an inhibitor of 5-HT synthesis
1966) also reduced morphine's
nucleus accumbens
1977).
in some regions of rat is unlikely, (Koe and
effect on DA metabolism
in the
(Spampinato et al., 1984) and there is evidence that
5-HT antagonists block the inhibitory action of 5-HT on dopamine-dependent
Pharmacological Research Communications, VoL 16, No. 5, 1984
516
effect Jn this area (Costall and Naylor, 1978). It has been shown recently that agents increasing 5-HT transmission reduce the morphine-induced increase of dopamine metabolism in the striatum of rats ($pampinato et al., 1983a).
This and other findings (Samanin et
al., 19~,~; Cervo et al., 1981; Spampinato et al., 1983b) have led to the suggestion that a single, moderate dose (I0 mg/kg) of morphine activates dopamine°-containing neurons in the striatum and that 5-HT mechanisms act presynaptically to inhibit this.
Morphine injected in the ventral teg-
mental ~rea (VTA) of rats has been reported to activate dopamine-containing neurons ~[nnervating the nucleus accumbens and other telencephalic regions (Joyce and Iversen, 1979) and 5-HT may influence dopamine mechanisms in the VTA (Beart and McDonald, 1982).
It is possible therefore that part of
morphine's effect on DA metabolism in the nucleus accumbens depends on its ability t~ activate DA-containing neurons in the VTA, and 5-HT mechanisms activated by d-fenfluramine and CPP act by inhibiting this effect.
The
apparent p~xadox of 5-HT antagonists and agonists having the same effect is therefore solved considering that morphine-induced activation of 5-HT mechanisms on postsynaptic elements in the nucleus accumbens may contribute to the increase in HVA (Costall and Naylor, 1978; Pycock et al., 1978) whereas 5-HT mechanisms controlling presynaptic DA activity reduce morphine's ability to activate dopamine-containing neurons in the rat brain (Samanin et al., 1980; Cervo et al., 1981; Spampinato et al., 1983b). That agents increasing 5-HT transmission inhibit morphine's effect on mesolimbic dopamine-containing neurons is of particular interest in view of the recent finding that the ventral tegmental area and the nucleus accumbens are involved in the reinforcing effects of opiates in rats (Olds, 1982; Britt and Wise, 1983).
ACKNOWLEDGEMENTS
This work was partially supported by Centro Nazionale delle Ricerche in the
contextof the Progetto Finalizzato "Medicina Proventiva e Riabili-
tativa": Sottoprogetto SP 7 "Tossicodipendenza". The authors are greatly indebted to Giancarlo Bianchi and Marco Landi for measuring morphine in the brain of the various experimental groups.
Pharmacological Research Communications, Vol. 16, No. 5, 1984 REFERENCES Beart P.M. and McDonald D. (1982) J. Pharm.Pharmac. 34: 591-593. Britt M.D. and Wise R.A. (1983) Brain Res. 258: 105-108. Cervo L., Romandini S. and Samanin R. (1981) Life Sci. 29: 2585-2591. Costall B., Fortune D.H. and Naylor R.J. (1977). Br. J. Pharmac. 60:266P. Costall B. and Naylor R.J. (1978) J. Pharm.Pharmac. 30: 257-259. Costall B., Naylor R.J., Marsden C.D. and Pycock C.J. (1976) J. Pharm. Pharmac. 28: 523-526. Garattini S., Caccia S., Mennini T., Samanin R., Consolo S. and Ladinsky H. (1979) Curr. med. Res. Opin. 6, suppl.l: 15-27. Haigler H.J. and Aghajanian G.K. (1977) Fed.Proc. 36: 2159-2164. Invernizzi R~, Cotecchia S., De Blasi A., Mennini T., Patacclni R. and Samanin R. (1981) Neurochem. Int. 3: 239-244. Invernizzi R. and Samanin R. (1981) Pharmac. Res. Commun. 13: 511-516. Joyce E.M.and Iversen S.D. (1979) Neurosei. Letters 14: 207-212. Koe B.K. and Weissman A. (1966) J. Pharmac. exp.Ther. 154: 449-517. Miranda F., Invernizzi R. and Samanin R. (1979) Pharmac. Res. Commun. ii: 455-466. Olds M.E. (1982) Brain Res. 237: 429-440. Pycock C.J., Horton R.W. and Carter C.J. (1978) Adv. Biochem.Psychopharmac. 19: 323-341. Rocchetti M. and Recchia M. (1982) Comput. Programs Biomed. 14: 7-20. Samanin R., Cervo L., Rochat C., Poggesi E. and Mennini T. (1980) Life Sci. 27: 1141-1146. Samanin R., Mennini T., Ferraris A., Bendotti C., Borsini F. and Garattini S. (1979) Naunyn Schmiedebergs' Arch. Pharmac. 308: 159-163. Snelgar R.S. and Vogt M. (1981) J. Physiol.(London) 314: 395-410. Spampinato U., Invernizzi R., Nowakowska E. and Samanin R. (1983a) Biochem. Pharmac. in press Spampinato U., Invernizzi R. and Samanin R. (1984) ~harmac. Res. Commun. This issue. SpamDinato U., Nowakowska E. and Samanin R. (1983b) J. Pharm. Pharmac. 35: 831-833. Todd R.D., Muldoon S.M. and Watson R.L. (1982) J. Chromatogr. 232: I01-ii0. Westerink B.H.C. and Korf J. (1976) Eur. J. Pharmac. 38: 281-291. Wightman R.M., Plotsky P.M., Strope E., Delcore R.Jr. and Adams R.N. (1977) Brain Res. 131: 345-349.
517