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Evidence of serotonin involvement in the effect of morphine on dopamine metabolism in the rat nucleus accumbens but not in the striatum
519
Pharmacological Research Communications, Vol. 16, No. 5, 1984
EVIDENCE OF SEROTONIN INVOLVEMENT IN THE EFFECT OF MORPHINE ON DOPAMINE METABOLISM IN THE RAT NUCLEUS ACCUMBENS BUT NOT IN THE STRIATUM
U. Spampinato, R. Invernizzi and R. Samanin °
Istituto di Ricerche Farmacologiche Via Eritrea 62,
"Mario Negri"
20157 MILAN, Italy
Received in final form 22 December 1983
SIn4MARY Morphine-induced
increase of dopamine metabolism in the striatum and
nucleus accumbens was studied in rats treated with parachlorophenylalanine, an inhibitor of serotonin synthesis, or metergoline and mianserin, serotonin antagonists.
two
Morphine's effect in the nucleus accumbens, but not
in the stria tum, was significantly reduced by all the agents used to reduce serotonin transmission.
It thus appears that part of the effect of morphine
on dopamine metabolism in the nucleus accumbens is mediated by its ability to activate 5-HT function in this area.
INTRODUCTION There is evidence of serotonin (5-HT) involvement in the effects of morphine on dopamine activity in the striatum and nucleus accumbens of rats. Parachlorophenylalanine
(PCPA), an inhibitor of 5-HT synthesis
(Koe and
Weissman, 1966), was recently found to prevent the inhibitory effect of morphine on amphetamine-induced
stereotypy (Cervo et al., 1981), an effect
commonly attributed to increased release of dopamine from nerve terminals in the striatum (Ernst, 1969; Costall et al., 1977a). were reported as blocking morphine-induced
5-HT antagonists
inhibition of hyperactivity
caused by dopamine injection in the nucleus accumbens
(Costall et al.,
1977b) suggesting that morphine-induced activation of 5-HT mechanisms acts by opposing dopamine activity on postsynaptic neuronal elements in this area.
°Correspondence
0031-6989/84/050519-05/$03,00/0
© 1984 The Italian Pharmacological Society
Pharmacological Research Communications, Vol. 16, No. 5. 1984
520
A single injection of morphine increased the metabolism of dopamine in the striatum and nucleus accumbens (Westerink and Korf, 1976) by a mechanism which is not completely clear. been poorly explored.
The role of serotonin in this effect has
There is one study showing that p-chloro-N-methyl-
amphetamine, an inhibitor of 5-HT synthesis (Miller et al., 1970) significantly increases morphine's effect on homovanillic acid (HVA) levels in rat. striatum (Groppe and Kuschinsky, 1975).
However, no information is available
on the effect of impairment of 5-HT function on morphine-induced changes of dopamine metabolism in the nucleus accumbens. In the present experiments the effects of morphine on dopamine metabolism in the striatum and nucleus accumbens were studied in rats treated with 5~HT antagonists or PCPA to see the extent to which 5-HT mechanisms are involved in morphine's effects.
MATERIALS AND METHODS Male CD-COBS rats (Charles River, Italy), 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 from 7 p.m.). They had free access to water and food.
The animals received iO mg/kg
morphine hydrochloride subcutaneously (Farmitalia-Carlo Erba, Milan, Italy) and were killed by decapitation i h later for biochemical assay.
Pretreat-
ments were as follows: i00 mg/kg PCPA base (Aldrich, Europe), suspended in carboxymethylcellulose (0.5%) was administered orally for three days and morphine was injected 48 h after the last dose.
I mg/kg metergoline base
(Farmitalia-Carlo Erba, Milan, Italy), dissolved in 1% ascorbic acid solution and 30 mg/kg mianserin hydrochloride (Ravasini Organon, Rome, Italy) dissolved in saline, were administered intraperitoneally respectively 3 h and 30 minutes before morphine.
The doses of PCFA, metergoline and
mianserin were chosen on the basis of previous studies showing their blockade of central 5-HT mechanisms (Koe and Weissman, 1966; Mawson and Whittington, 1970; Vargaftig et al., 1971). each treatment was used as controls.
The appropriate vehicle in
Immediately after decapitation, the
animals' striatum and nucleus accumbens were dissected and frozen on dry ice.
HVA was measured by high performance liquid chromatography with
electrochemical detection according to Wightman et al. (1977) with minor modifications (Invernizzi and Samanin, 1981).
The data were statistically
analyzed by ANOVA 2x2 (Rocchetti and Recchia, 1982).
F test for significant
Pharmaco/og~a/Resea~hCommun~atio~s, VoL 1~ No. ~ 1984
521
interaction was followed by Tukey's test to compare the experimental groups with their controls.
RESULTS As shown in Table I neither metergoline nor mianserin significantly modified HVA concentrations in the striatum and nucleus accumbens.
PCPA
significantly reduced striatal HVA but had no effect on HVA in the nucleus accumbens,
i0 mg/kg morphine significantly raised HVA concentrations in
both brain areas.
The effect of morphine in the nucleus accumbens was
significantly reduced by PCPA, metergoline and mianserin (F interaction p K 0.05) whereas none of these drugs significantly modified morphine's effect in the striatum.
DISCUSSION PCPA and 5-HT antagonists did not significantly change HVA concentrations in the nucleus accumbens indicating that at the doses used they did not affect dopamine activity in this area.
The effect of morphine on dopa-
mine metabolism in the nucleus accumbens was significantly reduced by treatment with PCPA or 5HT antagonists.
The fact that morphine's effect
in the striatum was not modified makes it unlikely that ~he drugs had interfered
with the entry of morphine in the brain.
The data suggest
that the effect of morphine in the nucleus accumbens, but not in the striatum, depends on its ability to activate 5-HT mechanisms in the brain. The inability of PCPA'or 5-HT antagonists to modify the effect of morphine in the striatum is in apparent contrast with results by Groppe and Ku~chinsky (1975) who found potentiation of morphine's effect in rats treated with p-chloro-N-methylamphetamine, a depletor of brain 5-HT (Miller et al., 1970).
Although we have no clear explanation for these discrepan-
cies, parachloroamphetamine's ability to affect brain catecholamines (Sanders-Bush et el., 1974) may have influenced Groppe and Kuschinsky's results. The differences between the striatum and the nucleus accumbens may he due to a different organization of 5-HT-DA interaction in this areas.
5-HT
injections in the nucleus accumbens inhibit the effect of locally applied dopamine (Costall et al., 1976), whereas stereotyped movements induced by high doses of apomorphine, an effect caused by direct activation of postsyr~ptic dopamine receptors in the striatum (Janssen et al., 1960; Ernst,
Pharmacofogica!Research Communications, Vo/. 16, No. 5, 1984
522
TABLE I Effect of morphine on HVA levels in the striatum and n.accLunbens of rats treated with p-chlorophenylalanine
(PCPA), metergoline or mianserin,
HVA (ng/g ~ S.E.)
Treatment (mg/kg) striatum
n.accumbens
Vehicle + saline
661 + 40
364 + 17
Vehicle + morphine (IO)
850 +
PCPA (lOOx3) + saline
504 + 40
PCPA (iOOx3) + morphine (IO)
687
Vehicle + saline
610 + 44
Vehicle + morphine (IO)
832 +
Metergoline
(I) + saline
654 + 29
485 + 15
Metergoline
(i) + morphine (I0)
950 + 54 n.s.
668 + 25 °
Vehicle + saline
622 + 30
381 + 16
Vehicle + morphine (iO)
961 +
Mianserin (30) + saline
523 + 20
421 + 19
Mianserin (30) + morphine (I0)
887 + 33 n.s.
735 + 42 °
48**
789 + 32** 326 + 16
+ 40 n.s.
608 + 32 ° 407 + 17
52**
733 + 33**
33**
916 + 61"*
Each value is the mean + S.E. of 6 animals. The last injection of PCPA (IOO mg/kg x 3 days), metergoline and mianserin were given respectively 48, 3 and 0.5 h before morphine. The animals were killed i h after morphine injection. * p ( O . O 5 ; ** p ~ O . O l compared with vehicle + saline . Tukey's test O p ~ O . O 5 (F interaction) n.s. : not significant ( p < 0 . O 5 F interaction).
1967; And~n et el., 1967) are little or not affected (Bendotti et el., 1980; Rotrosen et al., 1972) or may even be increased
(Pycock et al., 1978) by
agents which enhance 5-HT transmission, These data suggest that serotonin acts as a blocker of dopamine activity on postsynaptic neuronal elements in the nucleus accumbens, but not in the striatum.
Accordingly,
an injection of serotonin in the nucleus
accumbens has been found to increase dopamine metabolism in this area (Pycock et al., 1978), an effect seen after blockade of dopamine action on postsynaptic receptors
(Westerink and Korf, 1976).
It is likely therefore
that part of morphine's effect on dopamine metabolism in the nucleus
Pharmacological Research Communications, Vol. 16, No. 5, 1984 accumbens in mediated by its ability to activate 5-HT function on neurons located post-synaptically to the dopamine-containing neurons there.
.ACKNOWLEDGEMENTS
This study has been supported by Centro Nazionale delle Ricerche in the context of the Progetto Finalizzato "Medicina Preventiva e Riabilitativa": Sottoprogetto SP 7 "Tossicodipendenza".
REFERENCES
And~n N.E., Rubenson A., Fuxe K. and Hokfelt T. (1967) J. Pharm. Pharmae. 19: 627-629. Bendotti C., Borsini F., Zauini M.~., Samanin R. and Garattini S. (1980) Pharmac. Res. Commun. 12: 567-574. Cervo L., Romandini S. and Samanin R. (1981) Life Sci. 29: 2585-2591. Costall B., Fortune D.H. and Naylor R.J. (1977b) Br. J. Pharmac. 60: 266P-267P. Costall B., Naylor R.J., Cannon J.G. and Lee T. (1977a) J. Pharm. Pharmac. 29: 337-342. Costall B., Naylor R.J., Marsden C.D. and Pycock C.J. (1976) J. Pharm. Pharmac. 28: 523-526. Ernst A.M. (1967) Psychopha~nacologia IO: 316-323. Ernst A.M. (1969) Acta Physiol. Pharmac. N~erl. 15: 141-154. Groppe G. and Kuschinsky K. (1975) Neuropharmacology 14: 659-664. lnvernizzi R. and Samanin R. (1981) Pharmac. Res. Commun. 13: 511-516. Janssen P.A.J., Niemegeers C.J.C. and Jageneau A.H.M. (1960) ArzneimittelForsch. iO: 1003-1005. Koe B.K. and WeissmanA. (1966) J. Pharmac. Exp. Ther. 154: 499-516. Marwson C. and Whittington H. (1970) Br. J. Pharmac. 39: 223P. Miller F.P., Cox R.H.Jr., Snodgrass W.R. and Maickel R.P. (1970) Biochem. Pharmac. 19: 435-442. 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. Rotrosen J., Angrist B.M., Wallach M.B. and Gershon S. (1972) Eur. J. Pharmac. 20: 133-135. Sanders-Bush E., Gallager D.A. and Sulser F. (1974) Adv. Biochem. Psychopharmac. IO: 185-194. Vargaftig B.B., Coignet J.L., de Vos C.J., Grijsen H. and Bonta I.L. (1971) Eur. J. Pharmac. 16: 336-346. 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.
523
Pharmacological Research Communications, Vol. 16, No. 5, 1984
EVIDENCE OF SEROTONIN INVOLVEMENT IN THE EFFECT OF MORPHINE ON DOPAMINE METABOLISM IN THE RAT NUCLEUS ACCUMBENS BUT NOT IN THE STRIATUM
U. Spampinato, R. Invernizzi and R. Samanin °
Istituto di Ricerche Farmacologiche Via Eritrea 62,
"Mario Negri"
20157 MILAN, Italy
Received in final form 22 December 1983
SIn4MARY Morphine-induced
increase of dopamine metabolism in the striatum and
nucleus accumbens was studied in rats treated with parachlorophenylalanine, an inhibitor of serotonin synthesis, or metergoline and mianserin, serotonin antagonists.
two
Morphine's effect in the nucleus accumbens, but not
in the stria tum, was significantly reduced by all the agents used to reduce serotonin transmission.
It thus appears that part of the effect of morphine
on dopamine metabolism in the nucleus accumbens is mediated by its ability to activate 5-HT function in this area.
INTRODUCTION There is evidence of serotonin (5-HT) involvement in the effects of morphine on dopamine activity in the striatum and nucleus accumbens of rats. Parachlorophenylalanine
(PCPA), an inhibitor of 5-HT synthesis
(Koe and
Weissman, 1966), was recently found to prevent the inhibitory effect of morphine on amphetamine-induced
stereotypy (Cervo et al., 1981), an effect
commonly attributed to increased release of dopamine from nerve terminals in the striatum (Ernst, 1969; Costall et al., 1977a). were reported as blocking morphine-induced
5-HT antagonists
inhibition of hyperactivity
caused by dopamine injection in the nucleus accumbens
(Costall et al.,
1977b) suggesting that morphine-induced activation of 5-HT mechanisms acts by opposing dopamine activity on postsynaptic neuronal elements in this area.
°Correspondence
0031-6989/84/050519-05/$03,00/0
© 1984 The Italian Pharmacological Society
Pharmacological Research Communications, Vol. 16, No. 5. 1984
520
A single injection of morphine increased the metabolism of dopamine in the striatum and nucleus accumbens (Westerink and Korf, 1976) by a mechanism which is not completely clear. been poorly explored.
The role of serotonin in this effect has
There is one study showing that p-chloro-N-methyl-
amphetamine, an inhibitor of 5-HT synthesis (Miller et al., 1970) significantly increases morphine's effect on homovanillic acid (HVA) levels in rat. striatum (Groppe and Kuschinsky, 1975).
However, no information is available
on the effect of impairment of 5-HT function on morphine-induced changes of dopamine metabolism in the nucleus accumbens. In the present experiments the effects of morphine on dopamine metabolism in the striatum and nucleus accumbens were studied in rats treated with 5~HT antagonists or PCPA to see the extent to which 5-HT mechanisms are involved in morphine's effects.
MATERIALS AND METHODS Male CD-COBS rats (Charles River, Italy), 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 from 7 p.m.). They had free access to water and food.
The animals received iO mg/kg
morphine hydrochloride subcutaneously (Farmitalia-Carlo Erba, Milan, Italy) and were killed by decapitation i h later for biochemical assay.
Pretreat-
ments were as follows: i00 mg/kg PCPA base (Aldrich, Europe), suspended in carboxymethylcellulose (0.5%) was administered orally for three days and morphine was injected 48 h after the last dose.
I mg/kg metergoline base
(Farmitalia-Carlo Erba, Milan, Italy), dissolved in 1% ascorbic acid solution and 30 mg/kg mianserin hydrochloride (Ravasini Organon, Rome, Italy) dissolved in saline, were administered intraperitoneally respectively 3 h and 30 minutes before morphine.
The doses of PCFA, metergoline and
mianserin were chosen on the basis of previous studies showing their blockade of central 5-HT mechanisms (Koe and Weissman, 1966; Mawson and Whittington, 1970; Vargaftig et al., 1971). each treatment was used as controls.
The appropriate vehicle in
Immediately after decapitation, the
animals' striatum and nucleus accumbens were dissected and frozen on dry ice.
HVA was measured by high performance liquid chromatography with
electrochemical detection according to Wightman et al. (1977) with minor modifications (Invernizzi and Samanin, 1981).
The data were statistically
analyzed by ANOVA 2x2 (Rocchetti and Recchia, 1982).
F test for significant
Pharmaco/og~a/Resea~hCommun~atio~s, VoL 1~ No. ~ 1984
521
interaction was followed by Tukey's test to compare the experimental groups with their controls.
RESULTS As shown in Table I neither metergoline nor mianserin significantly modified HVA concentrations in the striatum and nucleus accumbens.
PCPA
significantly reduced striatal HVA but had no effect on HVA in the nucleus accumbens,
i0 mg/kg morphine significantly raised HVA concentrations in
both brain areas.
The effect of morphine in the nucleus accumbens was
significantly reduced by PCPA, metergoline and mianserin (F interaction p K 0.05) whereas none of these drugs significantly modified morphine's effect in the striatum.
DISCUSSION PCPA and 5-HT antagonists did not significantly change HVA concentrations in the nucleus accumbens indicating that at the doses used they did not affect dopamine activity in this area.
The effect of morphine on dopa-
mine metabolism in the nucleus accumbens was significantly reduced by treatment with PCPA or 5HT antagonists.
The fact that morphine's effect
in the striatum was not modified makes it unlikely that ~he drugs had interfered
with the entry of morphine in the brain.
The data suggest
that the effect of morphine in the nucleus accumbens, but not in the striatum, depends on its ability to activate 5-HT mechanisms in the brain. The inability of PCPA'or 5-HT antagonists to modify the effect of morphine in the striatum is in apparent contrast with results by Groppe and Ku~chinsky (1975) who found potentiation of morphine's effect in rats treated with p-chloro-N-methylamphetamine, a depletor of brain 5-HT (Miller et al., 1970).
Although we have no clear explanation for these discrepan-
cies, parachloroamphetamine's ability to affect brain catecholamines (Sanders-Bush et el., 1974) may have influenced Groppe and Kuschinsky's results. The differences between the striatum and the nucleus accumbens may he due to a different organization of 5-HT-DA interaction in this areas.
5-HT
injections in the nucleus accumbens inhibit the effect of locally applied dopamine (Costall et al., 1976), whereas stereotyped movements induced by high doses of apomorphine, an effect caused by direct activation of postsyr~ptic dopamine receptors in the striatum (Janssen et al., 1960; Ernst,
Pharmacofogica!Research Communications, Vo/. 16, No. 5, 1984
522
TABLE I Effect of morphine on HVA levels in the striatum and n.accLunbens of rats treated with p-chlorophenylalanine
(PCPA), metergoline or mianserin,
HVA (ng/g ~ S.E.)
Treatment (mg/kg) striatum
n.accumbens
Vehicle + saline
661 + 40
364 + 17
Vehicle + morphine (IO)
850 +
PCPA (lOOx3) + saline
504 + 40
PCPA (iOOx3) + morphine (IO)
687
Vehicle + saline
610 + 44
Vehicle + morphine (IO)
832 +
Metergoline
(I) + saline
654 + 29
485 + 15
Metergoline
(i) + morphine (I0)
950 + 54 n.s.
668 + 25 °
Vehicle + saline
622 + 30
381 + 16
Vehicle + morphine (iO)
961 +
Mianserin (30) + saline
523 + 20
421 + 19
Mianserin (30) + morphine (I0)
887 + 33 n.s.
735 + 42 °
48**
789 + 32** 326 + 16
+ 40 n.s.
608 + 32 ° 407 + 17
52**
733 + 33**
33**
916 + 61"*
Each value is the mean + S.E. of 6 animals. The last injection of PCPA (IOO mg/kg x 3 days), metergoline and mianserin were given respectively 48, 3 and 0.5 h before morphine. The animals were killed i h after morphine injection. * p ( O . O 5 ; ** p ~ O . O l compared with vehicle + saline . Tukey's test O p ~ O . O 5 (F interaction) n.s. : not significant ( p < 0 . O 5 F interaction).
1967; And~n et el., 1967) are little or not affected (Bendotti et el., 1980; Rotrosen et al., 1972) or may even be increased
(Pycock et al., 1978) by
agents which enhance 5-HT transmission, These data suggest that serotonin acts as a blocker of dopamine activity on postsynaptic neuronal elements in the nucleus accumbens, but not in the striatum.
Accordingly,
an injection of serotonin in the nucleus
accumbens has been found to increase dopamine metabolism in this area (Pycock et al., 1978), an effect seen after blockade of dopamine action on postsynaptic receptors
(Westerink and Korf, 1976).
It is likely therefore
that part of morphine's effect on dopamine metabolism in the nucleus
Pharmacological Research Communications, Vol. 16, No. 5, 1984 accumbens in mediated by its ability to activate 5-HT function on neurons located post-synaptically to the dopamine-containing neurons there.
.ACKNOWLEDGEMENTS
This study has been supported by Centro Nazionale delle Ricerche in the context of the Progetto Finalizzato "Medicina Preventiva e Riabilitativa": Sottoprogetto SP 7 "Tossicodipendenza".
REFERENCES
And~n N.E., Rubenson A., Fuxe K. and Hokfelt T. (1967) J. Pharm. Pharmae. 19: 627-629. Bendotti C., Borsini F., Zauini M.~., Samanin R. and Garattini S. (1980) Pharmac. Res. Commun. 12: 567-574. Cervo L., Romandini S. and Samanin R. (1981) Life Sci. 29: 2585-2591. Costall B., Fortune D.H. and Naylor R.J. (1977b) Br. J. Pharmac. 60: 266P-267P. Costall B., Naylor R.J., Cannon J.G. and Lee T. (1977a) J. Pharm. Pharmac. 29: 337-342. Costall B., Naylor R.J., Marsden C.D. and Pycock C.J. (1976) J. Pharm. Pharmac. 28: 523-526. Ernst A.M. (1967) Psychopha~nacologia IO: 316-323. Ernst A.M. (1969) Acta Physiol. Pharmac. N~erl. 15: 141-154. Groppe G. and Kuschinsky K. (1975) Neuropharmacology 14: 659-664. lnvernizzi R. and Samanin R. (1981) Pharmac. Res. Commun. 13: 511-516. Janssen P.A.J., Niemegeers C.J.C. and Jageneau A.H.M. (1960) ArzneimittelForsch. iO: 1003-1005. Koe B.K. and WeissmanA. (1966) J. Pharmac. Exp. Ther. 154: 499-516. Marwson C. and Whittington H. (1970) Br. J. Pharmac. 39: 223P. Miller F.P., Cox R.H.Jr., Snodgrass W.R. and Maickel R.P. (1970) Biochem. Pharmac. 19: 435-442. 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. Rotrosen J., Angrist B.M., Wallach M.B. and Gershon S. (1972) Eur. J. Pharmac. 20: 133-135. Sanders-Bush E., Gallager D.A. and Sulser F. (1974) Adv. Biochem. Psychopharmac. IO: 185-194. Vargaftig B.B., Coignet J.L., de Vos C.J., Grijsen H. and Bonta I.L. (1971) Eur. J. Pharmac. 16: 336-346. 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.
523