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Changes of serotonin and dopamine metabolism in various forebrain areas of rats injected with morphine either systemically or in the raphe nuclei dorsalis and medianus
Brain Research, 328 (1985) 89-95 Elsevier
89
BRE 10551
Changes of Serotonin and Dopamine Metabolism in Various Forebrain Areas of Rats Injected with Morphine Either Systemically or in the Raphe Nuclei Dorsalis and Medianus U. SPAMPINATO, E. ESPOSITO, S. ROMANDINI and R. SAMANIN lstituto di Ricerche Farmacologiche 'Mario Negri'. Via Eritrea 62. 20157 Milan (ltalv)
(Accepted May 29th, 1984) Key words: morphine - - raphe dorsalis - - raphe medianus - - serotonin metabolism - - dopamine metabolism
The regional brain metabolism of serotonin (5-HT) and dopamine (DA) was studied in rats injected with morphine either systemically or in the nuclei raphe medianus (MR) or dorsalis (DR). A subcutaneous injection of 10 mg/kg morphine significantly raised the levels of 5-hydroxyindoleacetic acid (5-HIAA) in the diencephalon, striatum, nucleus accumbens and cortex with no effect in the hippocampus. Similar changes in 5-HT metabolism were found in animals injected with 5 ug/0.5 ul in the DR whereas morphine injected in the MR raised 5-HIAA levels only in the nucleus accumbens. A subcutaneous or direct injection of morphine in the DR significantly raised the levels of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in the striatum and nucleus accumbens, but injection in the MR was ineffective. All the effects of morphine were blocked by naloxone, injected either intraperitoneally ( I mg/kg) or directly in the raphe nuclei (2/tg/0.5 ul). Pretreatment with parachlorophenylalanine, an inhibitor of serotonin synthesis, significantly reduced the effect of morphine injected in the DR on dopamine metabolism in the striatum and nucleus accumbens. The data suggest that a major mechanism by which morphine increases 5-HT metabolism in the rat forebrain is activation of 5-HT cells in the nucleus raphe dorsalis, and this action may contribute to the increased DA metabolism found in the animal injected with morphine in this brain area. INTRODUCTION The hypothesis that serotonin (5-HT) in the central nervous system is involved in the actions of morphine 16.25-27,32 is supported by the finding that the drug enhances the activity of 5-HT-containing neurons. Systemic administration of morphine enhances 5-HT metabolism in various brain areas and spinal cord 18,19,25.28,33, but the exact mechanism of this effect has been poorly investigated. Recently Vasko and Vogt 31 found that morphine injected in the striaturn increased the turnover of 5-HT in this area, whereas morphine injected in the region of the nucleus raphe magnus increased 5-HT turnover in the posterior medulla and spinal cord. These findings suggest that morphine may increase 5-HT activity by acting either on 5-HT-containing nerve terminals (striaturn) or cell bodies (nucleus raphe magnus). Recently, Forchetti et al. s found an increase in the levels of 5-hydroxyindoleacetic acid ( 5 - H I A A ) in the hippo-
campus of rats injected with 2D-Ala-metenkephalinamide in the nucleus raphe medianus, suggesting that opioid-sensitive sites act by enhancing 5-HT activity in this area. Most serotonergic innervation in the forebrain, including the striatum, originates in the nucleus raphe dorsalis (DR)-" but it is not known whether morphine increases 5-HT metabolism in forebrain areas through action on nerve terminals, as shown by Vasko and Vogt 31 in the striatum, or 5-HT cells in the DR. The present study was aimed at clarifying this by injecting morphine systemically or directly in the D R and measuring 5-HT metabolism in various forebrain areas. The effect of morphine injected in the MR on 5-HT metabolism was assessed as well. Systemic administration of morphine increases the metabolism of dopamine in the striatum and nucleus accumbens~5. 34.36 and it has been suggested that part of this effect may be due to morphine's ability to increase 5-HT transmission in these areas ~,3~. It was therefore of in-
Correspondence: R. Samanin, lstituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.
0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V.
90 terest to see whether m o r p h i n e injected in the raphe nuclei caused changes in d o p a m i n e metabolism in the striatum and nucleus accumbens. The effects were also studied in animals treated with p a r a c h l o r o p h e nylalanine ( P C P A ) , an inhibitor of 5-HT synthesis 13 to see whether i m p a i r m e n t of 5-HT transmission prevented the effect of m o r p h i n e on d o p a m i n e m e t a b o lism. MATERIALS AND METHODS Male C D - C O B S rats (Charles River, Italy), weighing 175-200 g, were used. T h e y were kept at constant r o o m t e m p e r a t u r e (21 + 1 °C) and relative humidity (60%) with a 12-h l i g h t - d a r k cycle (dark from 19.00 h). They had free access to water and food. In one experiment, 10 mg/kg m o r p h i n e hydrochloride (Salars, Como, Italy), calculated as free base, was dissolved in saline and injected subcutaneously into rats. The animals were killed by decapitation 1 h after injection. Naloxone hydrochloride (1 mg/kg) ( E n d o L a b o r a t o r i e s Inc., G a r d e n City, New York, U . S . A . ) was dissolved in saline and injected intraperitoneally 10 min before morphine. in another experiment, guide-cannulas m a d e of 0.65 m m diameter stainless-steel tubing, fixed in plexiglass holders, were i m p l a n t e d using a Stoelting stereotaxic instrument, under ethyl ether anesthesia, so that the lower tips were 2 m m above the injection sites. The following coordinates were used: M R A = 0.4, L = 0, D = - 2 . 6 ; D R A = 0.3, L = 0, D = -0.614. Stainless-steel stylets, 0.3 m m in d i a m e t e r and as long as the guide, kept the guides p a t e n t until the animals were given intracerebral injections 5 days later. A n i m a l s were accustomed to handling and
on the day of the test the stylets were withdrawn and replaced by injection units (0.3 d i a m e t e r stainless tubing) terminating 2 mm below the tip of the guides. M o r p h i n e hydrochloride (5 ~g) calculated as free base, was delivered in 0.5 ktl saline at a rate of (I.5 M/min through an A g l a m i c r o m e t e r syringe coupled to the injection unit. The dose of 5 ktg/0.5/~1 morphine was chosen, as it has been previously found to have functional effects when injected in brain tissue 37. After the injection, the cannula was left in situ for another 15 s to allow diffusion of the solution away from the injection cannula tip. The animals were killed by decapitation 1 h after morphine injection. Naloxone hydrochloride (2 ktg), dissolved in 0.5 gl saline, was administered using an additional injection assembly immediately before morphine. The location of the cannulas was d e t e r m i n e d histologically after the experiments. Only data from rats in which the cannulas were exactly placed in the M R or D R were included in the results. One group of rats received 100 mg/kg P C P A base (Aldrich, E u r o p e ) , suspended in 0.5% carboxymethylcellulose, orally for 3 days. M o r p h i n e (5/~g) was injected into the D R 48 h after the last dose of P C P A according to the p r o c e d u r e described above. In each experiment the a p p r o p r i a t e vehicle was used as control. Brains were r e m o v e d and the following forebrain areas were dissected according to the technique described by Glowinski and Iverseng: diencephalon, nucleus accumbens, corpus striatum, hippocampus and the remaining telencephalon (cortex). The tissues were immediately frozen on dry-ice. 5 - H I A A , homovanillic acid ( H V A ) and dihydroxyphenytacetic acid ( D O P A C ) were m e a s u r e d by high-performance liquid c h r o m a t o g r a p h y with electrochemical de-
TABLE I Effect of morphine on 5-HIAA in rat forebrain areas
Each value is the mean + S.E. of 7 animals. Naloxone was injected intraperitoneally 10 min before morphine and the animals were killed 1 h after morphine, n.s., not significant (P > 0.05 Student's t-test). Treatment (mg/kg)
Saline + saline Saline + morphine 1° Naloxone I + saline Naloxone I + morphine 1°
5-HIAA (ng/g + S.E.) Diencephalon
Striatum
N. accumbens
Hippocampus
Cortex
344 _+20 486 _+ 19" 347 _+24 328 _+ 14"*
319 + 28 504 _+ 19" 381 _+26 398 + 28**
338 _+28 558 + 16" 403 + 21 438 + 30**
208 _+9 231 + 36 n.s. -
216 + 7 319 _+ 13" 223 + 8 224 _+7**
* P < 0.01 compared with saline + saline. Tukey's test. ** P < 0.05, Finteraction.
91 TABLE II Effect of morphine injected in the raphe dorsalis (DR) or raphe medianus (MR) on 5-HIAA in rat forebrain areas Each value is the mean _+ S.E. of 7 animals. Naloxone was injected in the DR or MR immediately before morphine and the animals were killed 1 h after morphine, n.s., not significant (P > 0.(/5 Student's t-test). Treatment (ug/O.5 ul)
5-H1AA (ng/g +_S. E. ) Diencephalon
Striatum
N. accumbens
Hippocampus
Cortex
DR Saline + saline Saline + morphine5 Naloxone 2 + saline Naloxone 2 + morphine5
291 405 274 286
295 506 282 308
277 392 263 284
_+ 7 + 6** + 7 + 5***
215 +__13 229 _+ 17 n.s. -
220 _+ 7 3311 +_ 5** 210 _+ 8 237 +_ 8***
MR Saline + saline Saline + morphine5 Naloxone 2 + saline Naloxone2 + morphine5
262 _+ 22 256 + 14 n.s. -
325 391 312 331
+ 20 + 12" + 12 +_ 11"**
182 + 15 189 + 10 n.s. -
260 _+ 18 281 + 16 n.s. -
_+ 12 + 8** +_ 10 _+ 12"**
_+ 11 + 9** +_ 8 + 13"**
253 _+ 15 259 + 8 n.s. -
* P < 0.05 compared with saline + saline. Tukey's test. ** P < 0.01 compared with saline + saline. Tukey's test. *** P < 0.05, Finteraction. t e c t i o n a c c o r d i n g t o t h e m e t h o d o f W i g h t m a n et al. 35
m o r p h i n e on regional forebrain 5 - H I A A was com-
with m i n o r m o d i f i c a t i o n s l l . 5 - H T was d e t e r m i n e d us-
p l e t e l y b l o c k e d by 1 m g / k g n a l o x o n e ( P < 0.05, F in-
ing t h e m e t h o d o f P o n z i o a n d J o n s s o n 21. D A was
t e r a c t i o n ) . T a b l e II s h o w s t h e e f f e c t o f 5 /~g m o r -
m e a s u r e d a c c o r d i n g to t h e m e t h o d o f K e l l e r et a1.12.
p h i n e i n j e c t e d in t h e D R o r M R o n 5 - H 1 A A c o n c e n -
T h e d a t a w e r e statistically a n a l y z e d by A N O V A 2 x
t r a t i o n s in t h e d i f f e r e n t b r a i n r e g i o n s . M o r p h i n e in-
223 . F - t e s t f o r significant i n t e r a c t i o n was f o l l o w e d by
j e c t e d in t h e D R s i g n i f i c a n t l y r a i s e d 5 - H I A A levels
T u k e y ' s test to c o m p a r e t h e e x p e r i m e n t a l g r o u p s
in t h e d i e n c e p h a l o n , s t r i a t u m , n u c l e u s a c c u m b e n s
with c o n t r o l s . In s o m e e x p e r i m e n t s S t u d e n t ' s t-test
a n d c o r t e x , with n o e f f e c t in t h e h i p p o c a m p u s . M o r -
was u s e d as well.
p h i n e i n j e c t e d in t h e M R r a i s e d 5 - H I A A levels only in t h e n u c l e u s a c c u m b e n s . T h e e f f e c t s o f m o r p h i n e
RESULTS
i n j e c t e d in e i t h e r t h e D R o r M R w e r e b l o c k e d by a local i n j e c t i o n o f 2/~g n a l o x o n e ( P < 0.05, F i n t e r a c -
A s s h o w n in T a b l e I, 10 m g / k g m o r p h i n e s.c. significantly r a i s e d 5 - H I A A levels in t h e d i e n c e p h a l o n ,
tion). Subcutaneous
i n j e c t i o n o f 10 m g / k g m o r p h i n e
striatum, nucleus a c c u m b e n s and cortex, w h e r e a s no
raised H V A a n d D O P A C c o n c e n t r a t i o n s in t h e stria-
effect was f o u n d in t h e h i p p o c a m p u s . T h e e f f e c t o f
turn a n d
nucleus accumbens and
t h e e f f e c t was
TABLE III Effect of morphine on DOPA C and HVA in the rat striatum and n. accumbens Each value is the mean (ng/g) _+ S.E. of 7 animals. Naloxone was injected intraperitoneally 111min before morphine, and the animals were killed 1 h after morphine. Treatment (mg/kg)
Striatum DOPAC
HVA
DOPA C
HVA
Saline + saline Saline + morphinO ~ Naloxone 1 + saline Naloxone I + morphine m
458 733 462 472
312 662 368 422
481 876 526 624
380 924 422 566
_+ 31 +_ 31" + 30 + 15"*
N. accumbens
* P < 0.01 compared with saline + saline, Tukey's test. ** P < 0.05, Finteraction.
+_ 15 + 38* + 21 + 32**
_+ 15 + 33* +_ 17 _+ 23**
+_ 31 __+43* +_ 13 +_ 46**
92 TABLE IV Effect of morphine injected in the raphe dorsalis (DR) or raphe medianus (MR) on DOPAC and HVA in the rat striatum and n. accumbens
Each value is the mean (ng/g) _+ S.E. of 7 animals. Naloxone was injected in the DR or MR immediately before morphine and the animals were killed 1 h after morphine, n.s., not significant (P > 0.05, Student's t-test). Treatment (,ug/O.5;d)
DR Saline + saline Saline + morphine ~ Naloxone 2 + saline Naloxone 2 + morphine s MR Saline + saline Saline + morphine r
Striatum
N. accumbens
DOPA C
HVA
DOPA C
HVA
433 854 452 520
270 _+ 9 642 + 12" 280 + 9 342 _+ 10"*
467 769 452 519
357 746 368 423
348 + 12 318 _+_15 n.s.
502 +_ 21 522 + 31 n.s.
+ 15 + 19" _+ 9 _+ 11"*
403 +_ 20 385 _+ 16 n.s.
+ 12 + 10" _+ 20 _+ 8**
_+ 8 + 13" + 14 _+ 12"*
375 + 23 352 + 29 n.s.
* P < 0.01 compared with saline + saline. Tukey's test. ** P < 0.05, Finteraction. b l o c k e d by n a l o x o n e 1 m g / k g i.p. ( T a b l e III). H V A
slightly r e d u c e d in s t r i a t u m (controls 5718 + 249,
and D O P A C in the s t r i a t u m and nucleus a c c u m b e n s
P C P A 4977 + 179 ng/g + S . E . , P < 0.05, S t u d e n t ' s
w e r e e l e v a t e d after i n j e c t i o n of 5/~g m o r p h i n e in the
t-test) but w e r e n o t significantly m o d i f i e d in the n. ac-
D R but not in the M R , and the effect of the i n j e c t i o n
c u m b e n s (controls 4390 + 136, P C P A 3902 + 238
in the D R was p r e v e n t e d by 2 ~ g n a l o x o n e a p p l i e d di-
ng/g + S . E . , P > 0.05, S t u d e n t ' s t-test).
rectly in this a r e a ( P < 0.05, F i n t e r a c t i o n ) ( T a b l e IV).
DISCUSSION
T a b l e V shows the effect of P C P A on the increase of H V A and D O P A C levels in the s t r i a t u m and nu-
T h e p r e s e n t study indicates that a m a j o r m e c h a -
cleus a c c u m b e n s caused by m o r p h i n e i n j e c t e d in the
nism by which m o r p h i n e increases the m e t a b o l i s m of
D R . P C P A , which by itself did not m o d i f y H V A and
5 - H T in v a r i o u s f o r e b r a i n areas is a c t i v a t i o n of 5 - H T
DOPAC,
significantly r e d u c e d the effect of m o r -
n e u r o n s in the nucleus r a p h e dorsalis. T h a t m o r p h i n e
p h i n e on d o p a m i n e m e t a b o l i t e s ( P < 0.05, F interac-
lacks effect on 5 - H T m e t a b o l i s m in the h i p p o c a m p u s
tion). P C P A m a r k e d l y r e d u c e d the levels of s e r o t o -
is c o m p a t i b l e with the finding that m o s t 5 - H T inner-
nin in the s t r i a t u m (controls 274 _+ 7, P C P A 18 + 2
v a t i o n in this a r e a does not o r i g i n a t e in the nucleus
ng/g tissue, P < 0.01, S t u d e n t ' s t-test) and nucleus ac-
r a p h e dorsalis 2. A l t h o u g h the a n t a g o n i s m by nalox-
c u m b e n s (controls 476 + 14, P C P A 38 _+ 2 ng/g tis-
o n e clearly shows an i n v o l v e m e n t of o p i o i d sites, the
sue, P < 0.01, S t u d e n t ' s t-test). D A
exact m e c h a n i s m by which morphine" activates 5 - H T
levels w e r e
TABLE V Effect of PCPA on the DOPAC and HVA in the striatum and n. accumbens of rats injected with morphine in the raphe dorsalis (DR)
Each value is the mean (ng/g) + S.E. of 7 animals. The last injection of PCPA (100 mg/kg x 3 days) was given 48 h before morphine and the animals were killed 1 h after morphine. Treatment
Striatum
(mg/kg) + (ug/O.5 ul)
DOPAC
HVA
DOPAC
HVA
Vehicle + saline Vehicle + morphine~ PCPA (100 x 3) + saline PCPA (100 x 3) + morphine 5
466 788 483 531
327 619 348 416
459 833 467 618
360 + 758 + 400 + 528 +
+ 11 _+ 35* _+ 18 _+ 24**
* P < 0.01 compared with vehicle + saline. ** P < 0.05. Finteraction.
N. accumbens
_+ 10 + 12" + 10 _+ 15"*
+ 18 _+ 33* + 18 _+ 15"*
8 11" 19 28**
93 neurons is not clear. It is unlikely to be through direct action on 5-HT cells, since opiates have been shown to have no direct effects on 5-HT mechanisms such as uptake 7 or release 17. Moreover microiontophoretic administration of morphine has no effect on the firing of 5-HT cells in the DRL0. In view of the finding that opioid sites have predominantly inhibitory effects on neuronal systems in the brain 20-38. it is more likely that morphine activates 5-HT neurons by reducing the activity of neurons which inhibit 5-HT cells in the DR. A neuronal system containing gamma-aminobutyric acid ( G A B A ) as neurotransmitter has been described in the D R region 4 and it has been suggested that G A B A neurons act by inhibiting 5-HT cells in the D R 22. Consistent with this hypothesis is the fact that muscimol injected in the D R reduced the metabolism of 5-HT in the striatum 24, an area preferentially innervated by 5-HT neurons in the D R 2. Accordingly, it has been found that an injection of chlordiazepoxide in the D R reduces the release of 5-HT in the substantia nigra and D R through a mechanism which may involve increased G A B A transmission 29. It is likely, therefore, that morphine increases the activity of 5-HT cells in the D R by inhibiting G A B A neurons influencing 5-HT activity in this area. The same concentration of morphine which increased 5-HT metabolism in the D R was ineffective when applied in the M R with the exception of the nucleus accumbens, suggesting that the M R is much less sensitive to the action of morphine. This agrees with the fact that systemic administration of 10 mg/kg morphine (present results) or higher doses (20 mg/kg is, 30 mg/kg (our unpublished results)) does not change 5-HT metabolism in the hippocampus, an area preferentially innervated by 5-HT neurons originating in the MR 2. On the other hand, Forchetti et al. s reported an increase of hippocampal 5 - H I A A after injection of D-Ala-metenkephalinamide in the MR, indicating that the M R is not absolutely insensitive to opioids. To better explore this issue we injected 20 t~g/t~l morphine in the M R and again found no effect on hippocampal 5 - H I A A . It seems therefore that in our conditions MR is particularly insensitive to the action of locally applied morphine. In agreement with previous findings 15.34.3~', subcutaneous injection of 10 mg/kg morphine significantly raised H V A and D O P A C in the striatum and nucleus accumbens by a naloxone-dependent mechanism.
The same results were obtained after morphine injection in the D R but not in the MR. confirming that the DR is particularly sensitive to this drug's action. An increase in striatal dopamine metabolism was previously reported in rats injected with etorphine in the periacqueductal gray close to the DR region t . PCPA, an inhibitor of 5-HT synthesis t~. markedly reduced the effect of morphine on dopamine metabolites in the striatum and nucleus accumbens, suggesting 5-HT involvement. Although PCPA caused a slfght, but significant drop in striatal D A levels, it is unlikely that direct action on D A synthesis fully accounts for the reduced ability of morphine to increase D A metabolites in PCPA-treated animals. Besides the fact that D A levels in the nucleus accumbens and D A metabolites in the striatum and n. accumbens were not significantly affected by PCPA treatment, in a previous stud\ 4 we found that haloperidol and methylphenidate significantly raised H V A in animals pretreated with PCPA. exactly as in the present study. The lack of morphine effect cannot be attributed, therefore, to D A neurons being unable to increase the utilization of their transmitter after PCPA treatment. In agreement with the hypothesis that 5-HT mediates the effect of morphine on DA metabolites is the finding that intracerebral injection of 5,7-dihydroxytryptamme. a neurotoxin for 5-HTcontaining neurons ~. significantly reduced morphine's effect on rat striatal dopamine turnover% Additionally. Spampinato et al. ~' found that PCPA or 5-HT antagonists such as metergoline and mianserin reduced the effect of morphine on H V A in the nucleus accumbens. It seems therefore that part of morphine's effect on brain dopamine metabolism dcpends on its ability to activate serotonergic transmission. In conclusion, the data suggest that a major mechanism by which morphine increases 5-HT metabolism in the forebrain is through activation of 5-HT cells in the DR, and this effect may contribute to the increase of D A metabolism found in animals treated with morphine. ACKNOWLEDGEMENTS This study was partially supported by C N R Contract 82.02064.56. E.E. was supported by a Training Fellowship from Formez, Rome, Italy.
94 REFERENCES 1 Algeri, S.. Consolazione, A. and Plaznik, A.. Increased metabolism of dopamine and serotonin induced in forebrain areas by etorphine microinjection in periaqueductal gray, Europ. J. Pharmacol., 68 (1980) 383-384. 2 Azmitia, E. C., The serotonin-producing neurons of the midbrain median and dorsal raphe nuclei. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 9, Plenum Press, New York, 1978, pp. 233-314. 3 Baumgarten, H. G., Bj6rklund, A., Lachenmayer, L. and Nobin, A., Evaluation of the effects of 5,7-dihydroxytryptamine on serotonin and catecholamine neurons in the rat CNS, Acta physiol, scand., Suppl. 391 (1973) 2-17. 4 Belin, M. F., Aguera, M., Tappaz, M., McRae-Degueurce, A., Bobillier, P. and Pujol, J. F., GABA-accumulating neurons in the nucleus raphe dorsalis and periaqueductal gray in the rat: a biochemical and radioautographic study, Brain Research, 170 (1979) 279-297. 5 Crunelli, V., Bernasconi, S. and Samanin, R.. Effects of o-and L-fenfluramine on striatal homovanillic acid concentrations in rats after pharmacological manipulation of brain serotonin, Pharmacol. Res. Commun., 12 (1980) 215-223. 6 Demarest, K. T. and Moore, K. E., Disruption of 5-hydroxytryptaminergic neuronal function blocks the action of morphine on tuberoinfundibular dopaminergic neurons, Life Sci., 28 ( 1981) 1345-1351. 7 Donzanti, B. A. and Warwick, R. O., Effect of methadone and morphine on serotonin uptake in rat periaqueductal gray slices, Europ. J. Pharmacol., 59 (1979) 107-110. 8 Forchetti, C. M., Marco, E. J. and Meek, J. L., Serotonin and 7-aminobutyric acid turnover after injection into the median raphe of substance P and D-ala-met-enkephalin amide, J. Neurochem., 38 (1982) 1336-1341. 9 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain. 1. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 10 Haigler, H. G., Morphine: ability to block neuronal activity evoked by a nociceptive stimulus, Life Sci., 19 (1976) 841-858. 11 Invernizzi, R. and Samanin, R., Effects of metergoline on regional serotonin metabolism in the rat brain, Pharmacol. Res. Commun., 13 (1981) 511-516. 12 Keller, R., Oke, A., Mefford, I. and Adams, R. N., Liquid chromatographic analysis of catecholamines routine assay for regional brain mapping, Life Sci., 19 (1976) 995-1004. 13 Koe, B. K. and Weissman, A., p-Chlorophenylalanine: a specific depletor of brain serotonin, J. Pharmacol. exp. Ther., 154 (1966) 499-516. 14 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 15 Kuschinsky, K. and Hornykiewicz, O., Morphine catalepsy in the rat: relation to striatal dopamine metabolism, Europ. J. Pharmacol., 19 (1972) 119-122. 16 Major, C. T. and Pleuvry, B. J., Effects of a-methyl-p-tyrosine, p-chlorophenylalanine, L-/3-(3,4-dihydroxyphenyl)alanine, 5-hydroxytryptophan and diethyldithiocarbamate on the analgesic activity of morphine and methylamphetamine in the mouse, Brit. J. Pharmacol., 42 (1971) 512-521. 17 Mennini, T., Pataccini, R. and Samanin, R., Effects of narcotic analgesics on the uptake and release of 5-hydroxytryp-
tamine in rat synaptosomal preparations, Brit, J. Pharmacol., 64 (1978) 75-82. 18 Messing, R. B., Flinchbaugh, C. and Waymire, J. C., Tryptophan and 5-hydroxyindoles in different CNS regions following acute morphine, Europ. J. Pharmacol., 48 (1978) 137-140. 19 Miranda, F., Invernizzi, R. and Samanin. R., Studies on the mechanism of the interaction of narcotic analgesics with brain serotonin, Pharmacol. Res. Commun., 11 (1979) 455-466. 20 Nicoll, R. A., Alger, B. E. and Jahr, C. E., Enkephalin blocks inhibitory pathways in the vertebrate CNS, Nature (Lond.), 287 (1980) 22-25. 21 Ponzio, F. and Jonsson, G., A rapid and simple method for the determination of picogram levels of serotonin in brain tissue using liquid chromatography with electrochemical detection, J. Neurochem., 32 (1979) 129-132. 22 Przewlocka, B., Stala, L. and Scheel-Kriiger, J., Evidence that G A B A in the nucleus dorsalis raphe induces stimulation of locomotor activity and eating behavior, Life Sci., 25 (1979) 937-946. 23 Rocchetti, M. and Recchia, M., SPBS: statistical programs for biological sciences. Minicomputer software for applying routine biostatistical methods, Comput. Programs Biomed., 14 (1982) 7-20. 24 Romandini, S. and Samanin, R., Muscimol injections in the nucleus raphe dorsalis block the antinociceptive effect of morphine in rats. Apparent lack of serotonin involvement in muscimol's effect, Brit. J. Pharmacol., 81 (1984) 25-29. 25 Samanin, R., Miranda, F. and Mennini, T,, Serotonergic mechanisms of narcotic action. In M. L. Adler and L. Manara and R. Samanin (Eds.), Factors Affecting the Action of Narcotics, Raven Press, New York, 1978, pp. 523-541. 26 Samanin, R., Gumulka, W. and Valzelli, L., Reduced effects of morphine in midbrain raphe lesioned rats, Europ. J. Pharmacol., 10 (1970) 339-343. 27 Samanin, R. and Valzelli, L., Increase of morphine-induced analgesia by stimulation of the nucleus raphe dorsalis, Europ. J. Pharmacol., 16 (1971) 298-302. 28 Snelgar, R. S. and Vogt, M., Mapping, in the rat central nervous system, of morphine-induced changes in turnover of 5-hydroxytryptamine, J. Physiol. (Lond.). 314 (1981) 395-410. 29 Soubrie, P., Bias, C., Ferron, A. and Glowinski, J.. Chlordiazepoxide reduces in vivo serotonin release in the basal ganglia of enc6phale isol6 but not anesthetized cats: evidence for a dorsal raphe site of action, J. Pharmacol. exp. Ther., 226 (1983) 526-532. 30 Spampinato, U., Invernizzi, R. and Samanin, R., Evidence of serotonin involvement in the effect of morphine on dopamine metabolism in the rat nucleus accumbens but not in the striatum, Pharmacol. Res. Commun., 16 (1984) 519-523. 31 Vasko, M. R. and Vogt, M., Analgesia, development of tolerance, and 5-hydroxytryptamine turnover in the rat after cerebral and systemic administration of morphine, Neuroscience, 7 (1982) 1215-1225. 32 Way, E. L. and Shen, F.-H., Catecholamines and 5-hydroxytryptamine. In D. H. Clouet (Ed.), Narcotic Drugs. Biochemical Pharmacology, Plenum Press, New York. 1971. pp. 229-253. 33 Weil-Fugazza, J., Godefroy, F. and Besson, J.-M.. Changes in brain and spinal tryptophan and 5-hydroxyindoleacetic acid levels following acute morphine administration in normal and arthritic rats, Brain Research, 175 (1979) 291-301.
95 34 Westerink, B. H. C. and Korf, J,, Regional rat brain levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid: concurrent fluorometric measurement and influence of drugs, Europ. J. Pharrnacol., 38 (1976) 281-291. 35 Wightman, R. M., Plotsky, P. M., Strope, E., Delcore, R. Jr. and Adams, R. N., Liquid chromatographic monitoring ofCSF metabolites, Brain Research, 131 (1977) 345-349. 36 Wood, P. L., Stotland, W. M., Richard, J. W. and Rackham, A., Actions of Mu, Kappa, Sigma, Delta and agonist/
antagonist opiates on striatal dopaminergic function, J. Pharmacol. exp. Ther., 215 (1980) 697-703. 37 Yaksh, T. L. and Rudy, T. A., Narcotic analgesics CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 4 (1978) 299-359. 38 Zieglg~insberger, W., French, E. D., Siggins, G. R. and Bloom, F. E., Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons, Science, 205 (1979) 415-417.
89
BRE 10551
Changes of Serotonin and Dopamine Metabolism in Various Forebrain Areas of Rats Injected with Morphine Either Systemically or in the Raphe Nuclei Dorsalis and Medianus U. SPAMPINATO, E. ESPOSITO, S. ROMANDINI and R. SAMANIN lstituto di Ricerche Farmacologiche 'Mario Negri'. Via Eritrea 62. 20157 Milan (ltalv)
(Accepted May 29th, 1984) Key words: morphine - - raphe dorsalis - - raphe medianus - - serotonin metabolism - - dopamine metabolism
The regional brain metabolism of serotonin (5-HT) and dopamine (DA) was studied in rats injected with morphine either systemically or in the nuclei raphe medianus (MR) or dorsalis (DR). A subcutaneous injection of 10 mg/kg morphine significantly raised the levels of 5-hydroxyindoleacetic acid (5-HIAA) in the diencephalon, striatum, nucleus accumbens and cortex with no effect in the hippocampus. Similar changes in 5-HT metabolism were found in animals injected with 5 ug/0.5 ul in the DR whereas morphine injected in the MR raised 5-HIAA levels only in the nucleus accumbens. A subcutaneous or direct injection of morphine in the DR significantly raised the levels of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in the striatum and nucleus accumbens, but injection in the MR was ineffective. All the effects of morphine were blocked by naloxone, injected either intraperitoneally ( I mg/kg) or directly in the raphe nuclei (2/tg/0.5 ul). Pretreatment with parachlorophenylalanine, an inhibitor of serotonin synthesis, significantly reduced the effect of morphine injected in the DR on dopamine metabolism in the striatum and nucleus accumbens. The data suggest that a major mechanism by which morphine increases 5-HT metabolism in the rat forebrain is activation of 5-HT cells in the nucleus raphe dorsalis, and this action may contribute to the increased DA metabolism found in the animal injected with morphine in this brain area. INTRODUCTION The hypothesis that serotonin (5-HT) in the central nervous system is involved in the actions of morphine 16.25-27,32 is supported by the finding that the drug enhances the activity of 5-HT-containing neurons. Systemic administration of morphine enhances 5-HT metabolism in various brain areas and spinal cord 18,19,25.28,33, but the exact mechanism of this effect has been poorly investigated. Recently Vasko and Vogt 31 found that morphine injected in the striaturn increased the turnover of 5-HT in this area, whereas morphine injected in the region of the nucleus raphe magnus increased 5-HT turnover in the posterior medulla and spinal cord. These findings suggest that morphine may increase 5-HT activity by acting either on 5-HT-containing nerve terminals (striaturn) or cell bodies (nucleus raphe magnus). Recently, Forchetti et al. s found an increase in the levels of 5-hydroxyindoleacetic acid ( 5 - H I A A ) in the hippo-
campus of rats injected with 2D-Ala-metenkephalinamide in the nucleus raphe medianus, suggesting that opioid-sensitive sites act by enhancing 5-HT activity in this area. Most serotonergic innervation in the forebrain, including the striatum, originates in the nucleus raphe dorsalis (DR)-" but it is not known whether morphine increases 5-HT metabolism in forebrain areas through action on nerve terminals, as shown by Vasko and Vogt 31 in the striatum, or 5-HT cells in the DR. The present study was aimed at clarifying this by injecting morphine systemically or directly in the D R and measuring 5-HT metabolism in various forebrain areas. The effect of morphine injected in the MR on 5-HT metabolism was assessed as well. Systemic administration of morphine increases the metabolism of dopamine in the striatum and nucleus accumbens~5. 34.36 and it has been suggested that part of this effect may be due to morphine's ability to increase 5-HT transmission in these areas ~,3~. It was therefore of in-
Correspondence: R. Samanin, lstituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.
0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V.
90 terest to see whether m o r p h i n e injected in the raphe nuclei caused changes in d o p a m i n e metabolism in the striatum and nucleus accumbens. The effects were also studied in animals treated with p a r a c h l o r o p h e nylalanine ( P C P A ) , an inhibitor of 5-HT synthesis 13 to see whether i m p a i r m e n t of 5-HT transmission prevented the effect of m o r p h i n e on d o p a m i n e m e t a b o lism. MATERIALS AND METHODS Male C D - C O B S rats (Charles River, Italy), weighing 175-200 g, were used. T h e y were kept at constant r o o m t e m p e r a t u r e (21 + 1 °C) and relative humidity (60%) with a 12-h l i g h t - d a r k cycle (dark from 19.00 h). They had free access to water and food. In one experiment, 10 mg/kg m o r p h i n e hydrochloride (Salars, Como, Italy), calculated as free base, was dissolved in saline and injected subcutaneously into rats. The animals were killed by decapitation 1 h after injection. Naloxone hydrochloride (1 mg/kg) ( E n d o L a b o r a t o r i e s Inc., G a r d e n City, New York, U . S . A . ) was dissolved in saline and injected intraperitoneally 10 min before morphine. in another experiment, guide-cannulas m a d e of 0.65 m m diameter stainless-steel tubing, fixed in plexiglass holders, were i m p l a n t e d using a Stoelting stereotaxic instrument, under ethyl ether anesthesia, so that the lower tips were 2 m m above the injection sites. The following coordinates were used: M R A = 0.4, L = 0, D = - 2 . 6 ; D R A = 0.3, L = 0, D = -0.614. Stainless-steel stylets, 0.3 m m in d i a m e t e r and as long as the guide, kept the guides p a t e n t until the animals were given intracerebral injections 5 days later. A n i m a l s were accustomed to handling and
on the day of the test the stylets were withdrawn and replaced by injection units (0.3 d i a m e t e r stainless tubing) terminating 2 mm below the tip of the guides. M o r p h i n e hydrochloride (5 ~g) calculated as free base, was delivered in 0.5 ktl saline at a rate of (I.5 M/min through an A g l a m i c r o m e t e r syringe coupled to the injection unit. The dose of 5 ktg/0.5/~1 morphine was chosen, as it has been previously found to have functional effects when injected in brain tissue 37. After the injection, the cannula was left in situ for another 15 s to allow diffusion of the solution away from the injection cannula tip. The animals were killed by decapitation 1 h after morphine injection. Naloxone hydrochloride (2 ktg), dissolved in 0.5 gl saline, was administered using an additional injection assembly immediately before morphine. The location of the cannulas was d e t e r m i n e d histologically after the experiments. Only data from rats in which the cannulas were exactly placed in the M R or D R were included in the results. One group of rats received 100 mg/kg P C P A base (Aldrich, E u r o p e ) , suspended in 0.5% carboxymethylcellulose, orally for 3 days. M o r p h i n e (5/~g) was injected into the D R 48 h after the last dose of P C P A according to the p r o c e d u r e described above. In each experiment the a p p r o p r i a t e vehicle was used as control. Brains were r e m o v e d and the following forebrain areas were dissected according to the technique described by Glowinski and Iverseng: diencephalon, nucleus accumbens, corpus striatum, hippocampus and the remaining telencephalon (cortex). The tissues were immediately frozen on dry-ice. 5 - H I A A , homovanillic acid ( H V A ) and dihydroxyphenytacetic acid ( D O P A C ) were m e a s u r e d by high-performance liquid c h r o m a t o g r a p h y with electrochemical de-
TABLE I Effect of morphine on 5-HIAA in rat forebrain areas
Each value is the mean + S.E. of 7 animals. Naloxone was injected intraperitoneally 10 min before morphine and the animals were killed 1 h after morphine, n.s., not significant (P > 0.05 Student's t-test). Treatment (mg/kg)
Saline + saline Saline + morphine 1° Naloxone I + saline Naloxone I + morphine 1°
5-HIAA (ng/g + S.E.) Diencephalon
Striatum
N. accumbens
Hippocampus
Cortex
344 _+20 486 _+ 19" 347 _+24 328 _+ 14"*
319 + 28 504 _+ 19" 381 _+26 398 + 28**
338 _+28 558 + 16" 403 + 21 438 + 30**
208 _+9 231 + 36 n.s. -
216 + 7 319 _+ 13" 223 + 8 224 _+7**
* P < 0.01 compared with saline + saline. Tukey's test. ** P < 0.05, Finteraction.
91 TABLE II Effect of morphine injected in the raphe dorsalis (DR) or raphe medianus (MR) on 5-HIAA in rat forebrain areas Each value is the mean _+ S.E. of 7 animals. Naloxone was injected in the DR or MR immediately before morphine and the animals were killed 1 h after morphine, n.s., not significant (P > 0.(/5 Student's t-test). Treatment (ug/O.5 ul)
5-H1AA (ng/g +_S. E. ) Diencephalon
Striatum
N. accumbens
Hippocampus
Cortex
DR Saline + saline Saline + morphine5 Naloxone 2 + saline Naloxone 2 + morphine5
291 405 274 286
295 506 282 308
277 392 263 284
_+ 7 + 6** + 7 + 5***
215 +__13 229 _+ 17 n.s. -
220 _+ 7 3311 +_ 5** 210 _+ 8 237 +_ 8***
MR Saline + saline Saline + morphine5 Naloxone 2 + saline Naloxone2 + morphine5
262 _+ 22 256 + 14 n.s. -
325 391 312 331
+ 20 + 12" + 12 +_ 11"**
182 + 15 189 + 10 n.s. -
260 _+ 18 281 + 16 n.s. -
_+ 12 + 8** +_ 10 _+ 12"**
_+ 11 + 9** +_ 8 + 13"**
253 _+ 15 259 + 8 n.s. -
* P < 0.05 compared with saline + saline. Tukey's test. ** P < 0.01 compared with saline + saline. Tukey's test. *** P < 0.05, Finteraction. t e c t i o n a c c o r d i n g t o t h e m e t h o d o f W i g h t m a n et al. 35
m o r p h i n e on regional forebrain 5 - H I A A was com-
with m i n o r m o d i f i c a t i o n s l l . 5 - H T was d e t e r m i n e d us-
p l e t e l y b l o c k e d by 1 m g / k g n a l o x o n e ( P < 0.05, F in-
ing t h e m e t h o d o f P o n z i o a n d J o n s s o n 21. D A was
t e r a c t i o n ) . T a b l e II s h o w s t h e e f f e c t o f 5 /~g m o r -
m e a s u r e d a c c o r d i n g to t h e m e t h o d o f K e l l e r et a1.12.
p h i n e i n j e c t e d in t h e D R o r M R o n 5 - H 1 A A c o n c e n -
T h e d a t a w e r e statistically a n a l y z e d by A N O V A 2 x
t r a t i o n s in t h e d i f f e r e n t b r a i n r e g i o n s . M o r p h i n e in-
223 . F - t e s t f o r significant i n t e r a c t i o n was f o l l o w e d by
j e c t e d in t h e D R s i g n i f i c a n t l y r a i s e d 5 - H I A A levels
T u k e y ' s test to c o m p a r e t h e e x p e r i m e n t a l g r o u p s
in t h e d i e n c e p h a l o n , s t r i a t u m , n u c l e u s a c c u m b e n s
with c o n t r o l s . In s o m e e x p e r i m e n t s S t u d e n t ' s t-test
a n d c o r t e x , with n o e f f e c t in t h e h i p p o c a m p u s . M o r -
was u s e d as well.
p h i n e i n j e c t e d in t h e M R r a i s e d 5 - H I A A levels only in t h e n u c l e u s a c c u m b e n s . T h e e f f e c t s o f m o r p h i n e
RESULTS
i n j e c t e d in e i t h e r t h e D R o r M R w e r e b l o c k e d by a local i n j e c t i o n o f 2/~g n a l o x o n e ( P < 0.05, F i n t e r a c -
A s s h o w n in T a b l e I, 10 m g / k g m o r p h i n e s.c. significantly r a i s e d 5 - H I A A levels in t h e d i e n c e p h a l o n ,
tion). Subcutaneous
i n j e c t i o n o f 10 m g / k g m o r p h i n e
striatum, nucleus a c c u m b e n s and cortex, w h e r e a s no
raised H V A a n d D O P A C c o n c e n t r a t i o n s in t h e stria-
effect was f o u n d in t h e h i p p o c a m p u s . T h e e f f e c t o f
turn a n d
nucleus accumbens and
t h e e f f e c t was
TABLE III Effect of morphine on DOPA C and HVA in the rat striatum and n. accumbens Each value is the mean (ng/g) _+ S.E. of 7 animals. Naloxone was injected intraperitoneally 111min before morphine, and the animals were killed 1 h after morphine. Treatment (mg/kg)
Striatum DOPAC
HVA
DOPA C
HVA
Saline + saline Saline + morphinO ~ Naloxone 1 + saline Naloxone I + morphine m
458 733 462 472
312 662 368 422
481 876 526 624
380 924 422 566
_+ 31 +_ 31" + 30 + 15"*
N. accumbens
* P < 0.01 compared with saline + saline, Tukey's test. ** P < 0.05, Finteraction.
+_ 15 + 38* + 21 + 32**
_+ 15 + 33* +_ 17 _+ 23**
+_ 31 __+43* +_ 13 +_ 46**
92 TABLE IV Effect of morphine injected in the raphe dorsalis (DR) or raphe medianus (MR) on DOPAC and HVA in the rat striatum and n. accumbens
Each value is the mean (ng/g) _+ S.E. of 7 animals. Naloxone was injected in the DR or MR immediately before morphine and the animals were killed 1 h after morphine, n.s., not significant (P > 0.05, Student's t-test). Treatment (,ug/O.5;d)
DR Saline + saline Saline + morphine ~ Naloxone 2 + saline Naloxone 2 + morphine s MR Saline + saline Saline + morphine r
Striatum
N. accumbens
DOPA C
HVA
DOPA C
HVA
433 854 452 520
270 _+ 9 642 + 12" 280 + 9 342 _+ 10"*
467 769 452 519
357 746 368 423
348 + 12 318 _+_15 n.s.
502 +_ 21 522 + 31 n.s.
+ 15 + 19" _+ 9 _+ 11"*
403 +_ 20 385 _+ 16 n.s.
+ 12 + 10" _+ 20 _+ 8**
_+ 8 + 13" + 14 _+ 12"*
375 + 23 352 + 29 n.s.
* P < 0.01 compared with saline + saline. Tukey's test. ** P < 0.05, Finteraction. b l o c k e d by n a l o x o n e 1 m g / k g i.p. ( T a b l e III). H V A
slightly r e d u c e d in s t r i a t u m (controls 5718 + 249,
and D O P A C in the s t r i a t u m and nucleus a c c u m b e n s
P C P A 4977 + 179 ng/g + S . E . , P < 0.05, S t u d e n t ' s
w e r e e l e v a t e d after i n j e c t i o n of 5/~g m o r p h i n e in the
t-test) but w e r e n o t significantly m o d i f i e d in the n. ac-
D R but not in the M R , and the effect of the i n j e c t i o n
c u m b e n s (controls 4390 + 136, P C P A 3902 + 238
in the D R was p r e v e n t e d by 2 ~ g n a l o x o n e a p p l i e d di-
ng/g + S . E . , P > 0.05, S t u d e n t ' s t-test).
rectly in this a r e a ( P < 0.05, F i n t e r a c t i o n ) ( T a b l e IV).
DISCUSSION
T a b l e V shows the effect of P C P A on the increase of H V A and D O P A C levels in the s t r i a t u m and nu-
T h e p r e s e n t study indicates that a m a j o r m e c h a -
cleus a c c u m b e n s caused by m o r p h i n e i n j e c t e d in the
nism by which m o r p h i n e increases the m e t a b o l i s m of
D R . P C P A , which by itself did not m o d i f y H V A and
5 - H T in v a r i o u s f o r e b r a i n areas is a c t i v a t i o n of 5 - H T
DOPAC,
significantly r e d u c e d the effect of m o r -
n e u r o n s in the nucleus r a p h e dorsalis. T h a t m o r p h i n e
p h i n e on d o p a m i n e m e t a b o l i t e s ( P < 0.05, F interac-
lacks effect on 5 - H T m e t a b o l i s m in the h i p p o c a m p u s
tion). P C P A m a r k e d l y r e d u c e d the levels of s e r o t o -
is c o m p a t i b l e with the finding that m o s t 5 - H T inner-
nin in the s t r i a t u m (controls 274 _+ 7, P C P A 18 + 2
v a t i o n in this a r e a does not o r i g i n a t e in the nucleus
ng/g tissue, P < 0.01, S t u d e n t ' s t-test) and nucleus ac-
r a p h e dorsalis 2. A l t h o u g h the a n t a g o n i s m by nalox-
c u m b e n s (controls 476 + 14, P C P A 38 _+ 2 ng/g tis-
o n e clearly shows an i n v o l v e m e n t of o p i o i d sites, the
sue, P < 0.01, S t u d e n t ' s t-test). D A
exact m e c h a n i s m by which morphine" activates 5 - H T
levels w e r e
TABLE V Effect of PCPA on the DOPAC and HVA in the striatum and n. accumbens of rats injected with morphine in the raphe dorsalis (DR)
Each value is the mean (ng/g) + S.E. of 7 animals. The last injection of PCPA (100 mg/kg x 3 days) was given 48 h before morphine and the animals were killed 1 h after morphine. Treatment
Striatum
(mg/kg) + (ug/O.5 ul)
DOPAC
HVA
DOPAC
HVA
Vehicle + saline Vehicle + morphine~ PCPA (100 x 3) + saline PCPA (100 x 3) + morphine 5
466 788 483 531
327 619 348 416
459 833 467 618
360 + 758 + 400 + 528 +
+ 11 _+ 35* _+ 18 _+ 24**
* P < 0.01 compared with vehicle + saline. ** P < 0.05. Finteraction.
N. accumbens
_+ 10 + 12" + 10 _+ 15"*
+ 18 _+ 33* + 18 _+ 15"*
8 11" 19 28**
93 neurons is not clear. It is unlikely to be through direct action on 5-HT cells, since opiates have been shown to have no direct effects on 5-HT mechanisms such as uptake 7 or release 17. Moreover microiontophoretic administration of morphine has no effect on the firing of 5-HT cells in the DRL0. In view of the finding that opioid sites have predominantly inhibitory effects on neuronal systems in the brain 20-38. it is more likely that morphine activates 5-HT neurons by reducing the activity of neurons which inhibit 5-HT cells in the DR. A neuronal system containing gamma-aminobutyric acid ( G A B A ) as neurotransmitter has been described in the D R region 4 and it has been suggested that G A B A neurons act by inhibiting 5-HT cells in the D R 22. Consistent with this hypothesis is the fact that muscimol injected in the D R reduced the metabolism of 5-HT in the striatum 24, an area preferentially innervated by 5-HT neurons in the D R 2. Accordingly, it has been found that an injection of chlordiazepoxide in the D R reduces the release of 5-HT in the substantia nigra and D R through a mechanism which may involve increased G A B A transmission 29. It is likely, therefore, that morphine increases the activity of 5-HT cells in the D R by inhibiting G A B A neurons influencing 5-HT activity in this area. The same concentration of morphine which increased 5-HT metabolism in the D R was ineffective when applied in the M R with the exception of the nucleus accumbens, suggesting that the M R is much less sensitive to the action of morphine. This agrees with the fact that systemic administration of 10 mg/kg morphine (present results) or higher doses (20 mg/kg is, 30 mg/kg (our unpublished results)) does not change 5-HT metabolism in the hippocampus, an area preferentially innervated by 5-HT neurons originating in the MR 2. On the other hand, Forchetti et al. s reported an increase of hippocampal 5 - H I A A after injection of D-Ala-metenkephalinamide in the MR, indicating that the M R is not absolutely insensitive to opioids. To better explore this issue we injected 20 t~g/t~l morphine in the M R and again found no effect on hippocampal 5 - H I A A . It seems therefore that in our conditions MR is particularly insensitive to the action of locally applied morphine. In agreement with previous findings 15.34.3~', subcutaneous injection of 10 mg/kg morphine significantly raised H V A and D O P A C in the striatum and nucleus accumbens by a naloxone-dependent mechanism.
The same results were obtained after morphine injection in the D R but not in the MR. confirming that the DR is particularly sensitive to this drug's action. An increase in striatal dopamine metabolism was previously reported in rats injected with etorphine in the periacqueductal gray close to the DR region t . PCPA, an inhibitor of 5-HT synthesis t~. markedly reduced the effect of morphine on dopamine metabolites in the striatum and nucleus accumbens, suggesting 5-HT involvement. Although PCPA caused a slfght, but significant drop in striatal D A levels, it is unlikely that direct action on D A synthesis fully accounts for the reduced ability of morphine to increase D A metabolites in PCPA-treated animals. Besides the fact that D A levels in the nucleus accumbens and D A metabolites in the striatum and n. accumbens were not significantly affected by PCPA treatment, in a previous stud\ 4 we found that haloperidol and methylphenidate significantly raised H V A in animals pretreated with PCPA. exactly as in the present study. The lack of morphine effect cannot be attributed, therefore, to D A neurons being unable to increase the utilization of their transmitter after PCPA treatment. In agreement with the hypothesis that 5-HT mediates the effect of morphine on DA metabolites is the finding that intracerebral injection of 5,7-dihydroxytryptamme. a neurotoxin for 5-HTcontaining neurons ~. significantly reduced morphine's effect on rat striatal dopamine turnover% Additionally. Spampinato et al. ~' found that PCPA or 5-HT antagonists such as metergoline and mianserin reduced the effect of morphine on H V A in the nucleus accumbens. It seems therefore that part of morphine's effect on brain dopamine metabolism dcpends on its ability to activate serotonergic transmission. In conclusion, the data suggest that a major mechanism by which morphine increases 5-HT metabolism in the forebrain is through activation of 5-HT cells in the DR, and this effect may contribute to the increase of D A metabolism found in animals treated with morphine. ACKNOWLEDGEMENTS This study was partially supported by C N R Contract 82.02064.56. E.E. was supported by a Training Fellowship from Formez, Rome, Italy.
94 REFERENCES 1 Algeri, S.. Consolazione, A. and Plaznik, A.. Increased metabolism of dopamine and serotonin induced in forebrain areas by etorphine microinjection in periaqueductal gray, Europ. J. Pharmacol., 68 (1980) 383-384. 2 Azmitia, E. C., The serotonin-producing neurons of the midbrain median and dorsal raphe nuclei. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 9, Plenum Press, New York, 1978, pp. 233-314. 3 Baumgarten, H. G., Bj6rklund, A., Lachenmayer, L. and Nobin, A., Evaluation of the effects of 5,7-dihydroxytryptamine on serotonin and catecholamine neurons in the rat CNS, Acta physiol, scand., Suppl. 391 (1973) 2-17. 4 Belin, M. F., Aguera, M., Tappaz, M., McRae-Degueurce, A., Bobillier, P. and Pujol, J. F., GABA-accumulating neurons in the nucleus raphe dorsalis and periaqueductal gray in the rat: a biochemical and radioautographic study, Brain Research, 170 (1979) 279-297. 5 Crunelli, V., Bernasconi, S. and Samanin, R.. Effects of o-and L-fenfluramine on striatal homovanillic acid concentrations in rats after pharmacological manipulation of brain serotonin, Pharmacol. Res. Commun., 12 (1980) 215-223. 6 Demarest, K. T. and Moore, K. E., Disruption of 5-hydroxytryptaminergic neuronal function blocks the action of morphine on tuberoinfundibular dopaminergic neurons, Life Sci., 28 ( 1981) 1345-1351. 7 Donzanti, B. A. and Warwick, R. O., Effect of methadone and morphine on serotonin uptake in rat periaqueductal gray slices, Europ. J. Pharmacol., 59 (1979) 107-110. 8 Forchetti, C. M., Marco, E. J. and Meek, J. L., Serotonin and 7-aminobutyric acid turnover after injection into the median raphe of substance P and D-ala-met-enkephalin amide, J. Neurochem., 38 (1982) 1336-1341. 9 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain. 1. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 10 Haigler, H. G., Morphine: ability to block neuronal activity evoked by a nociceptive stimulus, Life Sci., 19 (1976) 841-858. 11 Invernizzi, R. and Samanin, R., Effects of metergoline on regional serotonin metabolism in the rat brain, Pharmacol. Res. Commun., 13 (1981) 511-516. 12 Keller, R., Oke, A., Mefford, I. and Adams, R. N., Liquid chromatographic analysis of catecholamines routine assay for regional brain mapping, Life Sci., 19 (1976) 995-1004. 13 Koe, B. K. and Weissman, A., p-Chlorophenylalanine: a specific depletor of brain serotonin, J. Pharmacol. exp. Ther., 154 (1966) 499-516. 14 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 15 Kuschinsky, K. and Hornykiewicz, O., Morphine catalepsy in the rat: relation to striatal dopamine metabolism, Europ. J. Pharmacol., 19 (1972) 119-122. 16 Major, C. T. and Pleuvry, B. J., Effects of a-methyl-p-tyrosine, p-chlorophenylalanine, L-/3-(3,4-dihydroxyphenyl)alanine, 5-hydroxytryptophan and diethyldithiocarbamate on the analgesic activity of morphine and methylamphetamine in the mouse, Brit. J. Pharmacol., 42 (1971) 512-521. 17 Mennini, T., Pataccini, R. and Samanin, R., Effects of narcotic analgesics on the uptake and release of 5-hydroxytryp-
tamine in rat synaptosomal preparations, Brit, J. Pharmacol., 64 (1978) 75-82. 18 Messing, R. B., Flinchbaugh, C. and Waymire, J. C., Tryptophan and 5-hydroxyindoles in different CNS regions following acute morphine, Europ. J. Pharmacol., 48 (1978) 137-140. 19 Miranda, F., Invernizzi, R. and Samanin. R., Studies on the mechanism of the interaction of narcotic analgesics with brain serotonin, Pharmacol. Res. Commun., 11 (1979) 455-466. 20 Nicoll, R. A., Alger, B. E. and Jahr, C. E., Enkephalin blocks inhibitory pathways in the vertebrate CNS, Nature (Lond.), 287 (1980) 22-25. 21 Ponzio, F. and Jonsson, G., A rapid and simple method for the determination of picogram levels of serotonin in brain tissue using liquid chromatography with electrochemical detection, J. Neurochem., 32 (1979) 129-132. 22 Przewlocka, B., Stala, L. and Scheel-Kriiger, J., Evidence that G A B A in the nucleus dorsalis raphe induces stimulation of locomotor activity and eating behavior, Life Sci., 25 (1979) 937-946. 23 Rocchetti, M. and Recchia, M., SPBS: statistical programs for biological sciences. Minicomputer software for applying routine biostatistical methods, Comput. Programs Biomed., 14 (1982) 7-20. 24 Romandini, S. and Samanin, R., Muscimol injections in the nucleus raphe dorsalis block the antinociceptive effect of morphine in rats. Apparent lack of serotonin involvement in muscimol's effect, Brit. J. Pharmacol., 81 (1984) 25-29. 25 Samanin, R., Miranda, F. and Mennini, T,, Serotonergic mechanisms of narcotic action. In M. L. Adler and L. Manara and R. Samanin (Eds.), Factors Affecting the Action of Narcotics, Raven Press, New York, 1978, pp. 523-541. 26 Samanin, R., Gumulka, W. and Valzelli, L., Reduced effects of morphine in midbrain raphe lesioned rats, Europ. J. Pharmacol., 10 (1970) 339-343. 27 Samanin, R. and Valzelli, L., Increase of morphine-induced analgesia by stimulation of the nucleus raphe dorsalis, Europ. J. Pharmacol., 16 (1971) 298-302. 28 Snelgar, R. S. and Vogt, M., Mapping, in the rat central nervous system, of morphine-induced changes in turnover of 5-hydroxytryptamine, J. Physiol. (Lond.). 314 (1981) 395-410. 29 Soubrie, P., Bias, C., Ferron, A. and Glowinski, J.. Chlordiazepoxide reduces in vivo serotonin release in the basal ganglia of enc6phale isol6 but not anesthetized cats: evidence for a dorsal raphe site of action, J. Pharmacol. exp. Ther., 226 (1983) 526-532. 30 Spampinato, U., Invernizzi, R. and Samanin, R., Evidence of serotonin involvement in the effect of morphine on dopamine metabolism in the rat nucleus accumbens but not in the striatum, Pharmacol. Res. Commun., 16 (1984) 519-523. 31 Vasko, M. R. and Vogt, M., Analgesia, development of tolerance, and 5-hydroxytryptamine turnover in the rat after cerebral and systemic administration of morphine, Neuroscience, 7 (1982) 1215-1225. 32 Way, E. L. and Shen, F.-H., Catecholamines and 5-hydroxytryptamine. In D. H. Clouet (Ed.), Narcotic Drugs. Biochemical Pharmacology, Plenum Press, New York. 1971. pp. 229-253. 33 Weil-Fugazza, J., Godefroy, F. and Besson, J.-M.. Changes in brain and spinal tryptophan and 5-hydroxyindoleacetic acid levels following acute morphine administration in normal and arthritic rats, Brain Research, 175 (1979) 291-301.
95 34 Westerink, B. H. C. and Korf, J,, Regional rat brain levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid: concurrent fluorometric measurement and influence of drugs, Europ. J. Pharrnacol., 38 (1976) 281-291. 35 Wightman, R. M., Plotsky, P. M., Strope, E., Delcore, R. Jr. and Adams, R. N., Liquid chromatographic monitoring ofCSF metabolites, Brain Research, 131 (1977) 345-349. 36 Wood, P. L., Stotland, W. M., Richard, J. W. and Rackham, A., Actions of Mu, Kappa, Sigma, Delta and agonist/
antagonist opiates on striatal dopaminergic function, J. Pharmacol. exp. Ther., 215 (1980) 697-703. 37 Yaksh, T. L. and Rudy, T. A., Narcotic analgesics CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain, 4 (1978) 299-359. 38 Zieglg~insberger, W., French, E. D., Siggins, G. R. and Bloom, F. E., Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons, Science, 205 (1979) 415-417.