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Effects of Morphine on Metabolism of Dopamine and Serotonin in Brains of Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats
Alcohol, Vol. 18, No. 1, pp. 3–10, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/99/$–see front matter
PII S0741-8329(98)00060-3
Effects of Morphine on Metabolism of Dopamine and Serotonin in Brains of Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats AAPO HONKANEN,*‡ PETRI HYYTIÄ,† ESA R. KORPI†‡ AND LIISA AHTEE* *Department of Pharmacy, Division of Pharmacology and Toxicology,University of Helsinki, POB 56, FIN-00014, University of Helsinki, Finland; †Department of Mental Health and Alcohol Research, National Public Health Institute, POB 719, FIN-00101, Helsinki, Finland; ‡Department of Pharmacology and Clinical Pharmacology, University of Turku, FIN-20520, Turku, Finland Received 22 April 1998; Accepted 6 October 1998 HONKANEN, A., P. HYYTIÄ, E. R. KORPI AND L. AHTEE. Effects of morphine on metabolism of dopamine and serotonin in brains of alcohol-preferring AA and alcohol-avoiding ANA rats. ALCOHOL 18(1) 3–10, 1999.—Morphine induces a larger locomotor stimulation in the alcohol-preferring AA rats than in the alcohol-avoiding ANA rats. We have now studied the acute effects of morphine (1 and 3 mg/kg) on metabolism of dopamine and serotonin (5-HT) in the dorsal and ventral striatum of the AA and ANA rats. The basal level of dopamine release, as reflected by the concentration of dopamine metabolite 3-methoxytyramine (3-MT), was lower in the caudate-putamen and nucleus accumbens of the AA rats than in the ANA rats. In the caudate-putamen, morphine increased dopamine metabolism and release more in the AA than in the ANA rats. In the nucleus accumbens and olfactory tubercle, the effects of morphine on dopamine metabolism and release did not differ between the rat lines. Morphine elevated the metabolism of 5-HT in the caudate-putamen and nucleus accumbens of the AA but not in those of the ANA rats. The results suggest that the larger morphine-induced psychomotor stimulation of the AA rats in comparison with the ANA rats is associated with the larger effect of morphine on dopamine metabolism in the caudate-putamen and 5-HT metabolism in the caudate-putamen and nucleus accumbens. Furthermore, low basal dopamine release may play a role in the high alcohol-preference of AA rats. © 1999 Elsevier Science Inc. All rights reserved. Alcohol preference Nucleus accumbens
Alcohol avoidance Caudate-Putamen
Dopamine Morphine 3-Methoxytyramine Olfactory tubercle Selected rat lines
tides (14) and dopamine (24) and also change acutely and/or chronically opioid (10,53) and dopamine (23) receptors in brain. Furthermore, blockade of opioid receptors antagonizes the stimulatory effects of alcohol on dopamine metabolism and release in experimental animals (1,48,56). These findings suggest that the effects of alcohol on dopaminergic mechanisms could be mediated by endogenous opioids; i.e., the alcohol-induced release of endogenous opioids could stimulate dopamine release similarly as exogenous opioids do. The in-
VARIOUS drugs of abuse are believed to cause their reinforcing effects, at least partly, by stimulating dopamine release in the nucleus accumbens through various primary mechanisms (33,58). For instance, m-opioid receptor agonists, such as heroin and morphine, increase activity of dopamine neurons by disinhibiting dopaminergic cells in the VTA (16,25), which leads to increased dopamine release in the nucleus accumbens (27,37). Alcohol is believed to increase the release of opioid pep-
Requests for reprints should be addressed to Aapo Honkanen, Department of Pharmacology and Clinical Pharmacology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Tel: 1358-2-333-7678; Fax: 1358-2-333-7000; E-mail: [email protected]
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volvement of brain opioidergic system in the regulation of alcohol drinking is supported by findings that blockade of opioid receptors decreases alcohol drinking in experimental animals and humans (18,60). Acceleration of dopamine release in the nucleus accumbens results also in psychomotor stimulation, which, for example in rats and mice, is seen as increased locomotor activity (36,58). In a recent study, we found that morphine, a relatively selective m-opioid receptor agonist, induces larger locomotor stimulation in alcohol-preferring AA (Alko, Alcohol) rats than in alcohol-avoiding ANA (Alko, Nonalcohol) or nonselected Wistar rats (21). Based on the theory of homology between psychomotor stimulation and reinforcement (58), this finding suggests that alcohol-preferring rats are hypersensitive to the reinforcing effects of opioids. Therefore, a possible mechanism underlying the high alcohol-preference of AA rats could be a strong alcohol-induced activation of opioid systems, which could, in turn, cause enhanced dopamine release in these rats. Alternatively, m-opioid receptor agonists may induce both locomotor stimulation and reinforcement via dopamine-independent mechanisms, e.g., by acting on nondopaminergic cells in the nucleus accumbens (11). The aim of the present study was to investigate whether the effects of morphine on metabolism and release of dopamine in the dorsal (caudate-putamen) and ventral striatum (nucleus accumbens and olfactory tubercle) differ in the AA and ANA rats. Morphine-induced changes in the caudateputamen were assessed because it is possible that motor effects of opioids are partially mediated by this brain region (2). The olfactory tubercle was included in the study because, in addition to the nucleus accumbens, dopaminergic mechanism of this brain regions may be involved in regulation of alcohol drinking (19,47). Previously, it has been shown that concentrations of serotonin (5-hydroxytryptamine, 5-HT) are larger in the brains of the AA rats than in the ANA rats (5) suggesting involvement of this neurotransmitter in the differential alcohol preference of these animals. Like dopamine, 5-HT mechanisms are regulated by opioids (32,52) and they are involved in the regulation of alcohol consumption both in experimental animals (40) and humans (38,57). Furthermore, 5-HT regulates dopaminergic neurons (7,42) and locomotor activity (13,15,42). Therefore, morphine effects on metabolism of 5-HT were also assessed in the AA and ANA rats. METHOD
Animals Three-months-old AA and ANA rats from generations F69 and F72 (Department of Mental Health and Alcohol Research, National Public Health Institute) were used. The animals were housed in stainless-steel wire-mesh cages in groups of 4–5 animals per cage. The animals were maintained on a 12-h dark/light cycle (lights on from 600 to 1800 h), and ambient temperature of 20 6 28C and the relative air humidity of 50 6 5%. R3 rat pellet food (Ewos AB, Södertälje, Sweden) and tap water were available ad libitum. Written permissions for all experiments were obtained from the experimental animal committee of the National Public Health Institute. Experimental Procedures In experiment 1, the animals (n 5 8–11 per group) were given saline (1 ml/kg, SC) or morphine hydrochloride (Ph. Eur) at the doses of 1 or 3 mg/kg (as base in saline, SC). The
animals were killed 60 min after injections with head-focused microwave irradiation by using the NJE 2603-10 kW microwave applicator (New Japan Radio Inc., Japan) set at output power of 7 kW and irradiation time of 1.4 s. Experiment 2 was carried out in order to get more information on the time course of the effects of morphine on dopamine metabolism. The animals were killed 120 min after drug administration, and due to limited availability of the AA and ANA rats, only one morphine dose, 1 mg/kg, was used. During the time period between experiments 1 and 2, we found that voluntary alcohol drinking increases dopamine release not only in the the nucleus accumbens, but also in the olfactory tubercle of the AA rats (19). Therefore, the latter brain region was included in experiment 2 as well. After killing, the animals were decapitated and their heads were allowed to cool in crushed ice for 60 s. Then the brains of the animals were removed from the skulls, placed in a zinc brain mold (RBM-4000C, ASI Instruments, USA) cooled on ice, and sectioned coronally with razor blades at 2.7, 20.3 and 24.3 mm from bregma (44). The caudate-putamen and the nucleus accumbens were dissected from the second slice as described earlier (20) and the tissue including olfactory tubercle in the second slice located ventral to the nucleus accumbens was also dissected. The tissues were immediately frozen on dry ice and stored at 2808C before assayed for dopamine, 5-HT and their metabolites. Estimation of Brain Monoamines and Their Metabolites Samples were homogenized in 1 ml of 0.2 M HClO4 after which 25 ml KOH/HCOOH buffer was added to the homogenates to adjust the pH to 2.4. Samples were centrifuged first at 5,500 3 g for 45 min and then at 28,000 3 g for 20 min. The supernatants were purified using the method described earlier (17). In brief, a 950 ml sample of supernatant was pipetted onto Sephadex G-10 columns and washed with 1.8 ml of 0.01 M HCl. Dopamine and 3-MT were collected with 1.7 ml 0.01 M HCl and 1.0 ml 0.02 M NH3. The acidic metabolites HVA and DOPAC were collected by washing the columns with 1.0 ml of 0.02 M NH3 and 1.0 ml of 0.01 M KOH. Twenty ml of 2.6 mM sodium pyrosulfite and 5.7 mM ascorbic acid were added into the tubes containing dopamine/3-MT and DOPAC/ HVA, respectively. The samples were assayed for the concentration of dopamine and its metabolites by using HPLC with electrochemical detection as described earlier (22). Statistics Differences in the basal concentrations of monoamines and their metabolites between the rat lines were tested with Student’s t-test with Bonferroni adjustment. Drug effects, rat line differences and interaction between rat line and drug effect were evaluated first by using two-way analysis of variance (2-way ANOVA) followed by the Tukey compromise posthoc test. Since the two experiments conducted differed in design, the data from these experiments were tested separately with ANOVA. RESULTS
Basal Concentrations of Dopamine, 5-HT and Their Metabolites In order to compare the basal concentrations of monoamines and their metabolites between the rat strains, the data from the control groups of the two experiments were combined and are presented in Table 1. Dopamine concentrations were not
MORPHINE EFFECTS IN AA AND ANA RATS
5 TABLE 1
THE MEAN BASAL CONCENTRATIONS (6 SE) OF DOPAMINE AND ITS METABOLITES [3,4-DIHYDROXYPHENYL ACETIC ACID (DOPAC), HOMOVANILLIC ACID (HVA) AND 3-METHOXYTYRAMINE (3-MT)] AND 5-HYDROXYTRYPTAMINE (5-HT) AND ITS METABOLITE 5-HYDROXYINDOLEACETIC ACID (5-HIAA) IN DIFFERENT BRAIN AREAS OF ALCOHOL-PREFERRING AA AND ALCOHOL-AVOIDING ANA RATS Concentration (ng/g) Dopamine
Caudate putamen AA ANA Nucleus Accumbens AA ANA Olfactory tubercle AA ANA
DOPAC
HVA
3-MT
5-HT
5-HIAA
620 6 30 450 6 25†
470 6 10 400 6 15
14700 6 310 11000 6 640†
920 6 40 900 6 70
750 6 30 720 6 40
12.4 6 0.5 15.0 6 0.8*
9900 6 410 9500 6 380
960 6 70 860 6 100
640 6 30 630 6 25
5.3 6 0.2 6.5 6 0.4†
1390 6 60 1210 6 70
610 6 20 540 6 20
6600 6 170 5700 6 340
670 6 40 630 6 50
430 6 20 420 6 30
6.2 6 0.4 6.5 6 0.2
1700 6 90 1460 6 70
400 6 30 360 6 10
*p , 0.05, †p , 0.01 in comparison with AA rats (Student’s t-test with Bonferroni adjustment, n 5 8–19).
estimated from samples of the first experiment, so the data shown is from the experiment 2 only, as well as the data from the olfactory tubercle, which was not studied in the first experiment. In the caudate-putamen, the concentration of 3-MT was smaller in the AA than in the ANA rats, but, as previously reported (3,4,5), the concentrations of dopamine, 5-HT and 5-HIAA were higher in AA rats than in the ANA rats. The concentrations of the DOPAC and HVA did not differ in this brain region between the rat lines. In the nucleus accumbens, the 3-MT concentration was again smaller in the AA rats than in the ANA rats, while other meausures did not differ significantly between the rat lines. In the olfactory tubercle, concentration of 5-HT tended to be higher in the AA rats than ANA rats, but the diference did not reach statistical significance. Morphine Effects 60 min After Treatment (Experiment 1) In the caudate-putamen, 2-way ANOVA showed a rat line, F(1, 56) 5 8.65, p , 0.01 and treatment effects, F (2, 56) 5 12.8, P , 0.001; but no rat x treatment interaction F (2, 56) 5 0.86, p 5 0.43 for 3-MT. Posthoc comparison, however, revealed that 3-MT levels were significantly elevated by both morphine doses in the AA rats but not in the ANAs (Fig. 1). For DOPAC, there were significant rat line, F (1, 56) 5 5.78, p , 0.05 and treatment effects, F (2, 56) 5 50.4, p , 0.001 as well as significant rat line x treatment interaction F (2, 56) 5 3.8, p , 0.05, which was apparently due to larger elevation of DOPAC levels in the AA rats than in the ANAs. Similarly, ANOVA showed a rat line, F (1, 56) 5 8.43, p , 0.01 and treatment effects, F (2, 56) 5 44.8, p , 0.001, and rat line x treatment interaction, F (2, 56) 5 3.29, p , 0.05 for HVA. Morphine-induced elevation of HVA concentrations was greater in the AA rats than in the ANA rats. Morphine did not alter 5-HT concentration in the caudateputamen: treatment effect, F (2, 56) 5 1.47, p 5 0.24, but there was a significant rat line effect, F (1, 56) 5 28.9, p , 0.001 and no interaction, F (2, 56) 5 0.83, p 5 0.44. For 5-HIAA, rat line and treatment effects were highly significant, F (1, 56) 5 43.6. p , 0.001 and F (2, 56) 5 10.7. p , 0.001, respectively, but no interaction between them were found, F (2, 56) 5 1.62, p 5 0.21. Posthoc test indicated that 5-HIAA levels were significantly elevated by morphine in the AA rats only.
In the nucleus accumbens, ANOVA showed significant rat line, F (1, 55) 5 6.74 p , 0.05, and treatment effects, F (2, 56) 5 23.7, p , 0.001, for 3-MT, but no rat line x treatment interaction, F (2, 56) 5 2.93, p 5 0.06. Significant treatment effects were found for DOPAC, F (2, 55) 5 15.6, p , 0.001, and HVA, F (2, 55) 5 81.4, p , 0.001, but no rat line effects, F (1, 55) 5 0.008, p 5 0.93 (for DOPAC) and F (1, 55) 5 1.21, p 5 0.28 (for HVA), or interactions were found F (2, 56) 5 1.45, p 5 0.25 (for DOPAC) and F (1, 55) 5 0.27, p 5 0.77 (for HVA). For 5-HT, ANOVA showed a significant rat line effect, F (1, 54) 5 9.87, p , 0.01, but no treatment effect, F (2, 54) 5 1.12, p 5 0.33, or interaction, F (2, 54) 5 2.55, p 5 0.088. For 5-HIAA, ANOVA showed highly significant rat line effect, F (1, 55) 5 36.2, p , 0.001, and treatment effect, F (2, 55) 5 8.78, p , 0.001, but no interaction between them, F (2, 55) 5 1.95, p 5 0.15. Posthoc test, however, showed that 5-HIAA levels were elevated in the AA rats but not in the ANAs. Morphine Effects 120 min After Treatment (Experiment 2) In the caudate-putamen, two h after the morphine treatment (1 mg/kg), significant treatment effects were found for DOPAC, F (1, 28) 5 8.59, p , 0.01; HVA, F (1, 28) 5 27.1, p , 0.001, and 5-HIAA, F (1, 28) 5 5.72, p , 0.05, but not for dopamine, F (1, 28) 5 0.56, p 5 0.46 and 3-MT, F (1, 25) 5 0.67, p 5 0.42, and no rat line x treatment interactions were found (Table 2). Significant rat line effects were found for dopamine, F (1,28) 5 20.0, p , 0.001; DOPAC, F (1,28) 5 8.59, p , 0.01; HVA, F (1,28) 5 6.14, p , 0.05; 5-HT, F (1,28) 5 12.9, p , 0.01 and 5-HIAA, F (1,28) 5 10.7, p , 0.01, but not for 3-MT. In posthoc comparisons only HVA concentrations were elevated in both rat lines. In the nucleus accumbens, the concentrations of dopamine, F (1, 28) 5 4.51, p , 0.05 and HVA, F (1, 28) 5 18.1, p , 0.001 were altered by morphine treament, but not those of 3-MT, F (1, 28) 5 2.05, P 5 0.16; DOPAC, F (1, 28) 5 2.56, p , 0.12 and 5-HIAA, F (1, 28) 5 0.22, p , 0.64 (Table 2). ANOVA showed a significant rat line effects for dopamine, F (1, 28) 5 5.10, p , 0.05; 5-HT, F (1, 28) 5 7.78, p , 0.01, and 5-HIAA, F (1,28) 5 10.1, p , 0.01, but not for other measures. Posthoc test showed that dopamine was significantly decreased by morphine in the ANA rats but not in the AA rats, and HVA was significantly increased in the AA rats only.
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FIG. 1. Effects of morphine (1 or 3 mg/kg, s.c.) or saline (SAL) on the dopamine metabolites, 3-methoxytyramine (3-MT), homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid DOPAC and on the 5-HT metabolite 5-hydroxyindolacetic acid (5-HIAA) in the caudate-putamen and in the nucleus accumbens of the AA and ANA (experiment 1). Rats were given morphine or 0.9 % NaCl (SAL) and killed 60 min later with head-focused microwave irradiation. *p , 0.05 and **p , 0.01, in comparison with respective SAL-treated control group (Tukey compromise test).
In the olfactory tubercle, ANOVA showed a significant treatment effects for 3-MT, F (1, 27) 5 10.9, p , 0.05; DOPAC, F (1, 27) 5 16.7, p , 0.01; HVA, F (1, 27) 5 40.5, p , 0.001, but not for dopamine, F (1, 27) 5 0.04, p 5 0.85; 5-HT, F (1, 27) 5 0.28, p , 0.60 or 5-HIAA, F (1, 27) 5 1.92, p , 0.18 (Table 2). A significant rat line x treatment interaction
was found only for 5-HIAA, F (1, 27) 5 5.28, p , 0.05. ANOVA showed a significant rat line effects for dopamine, F (1,27) 5 7.64, p , 0.05; for 5-HT, F (1,27) 5 13.8, p , 0.001 and for 5-HIAA, F (1,27) 5 14.5, p , 0.001. Posthoc test showed that DOPAC and HVA concentrations were elevated in both rat lines, but 5-HIAA only in the AA rats.
MORPHINE EFFECTS IN AA AND ANA RATS
7 TABLE 2
EFFECTS OF MORPHINE (MO, 1 mg/kg, SC) ON THE CONCENTRATIONS DOPAMINE AND ITS METABOLITES [3,4-DIHYDROXYPHENYL ACETIC ACID (DOPAC), HOMOVANILLIC ACID (HVA) AND 3-METHOXYTYRAMINE (3-MT)] AND 5-HYDROXYTRYPTAMINE (5-HT) AND ITS METABOLITE 5-HYDROXYINDOLACETIC ACID (5-HIAA) IN DIFFERENT BRAIN AREAS OF ALCOHOL-PREFERRING AA AND ALCOHOL-AVOIDING ANA RATS 120 MIN AFTER INJECTIONS (EXPERIMENT 2) Concentration (ng/g)
Caudate-Putamen AA Saline Mo ANA Sal Mo Nucleus accumbens AA Saline Mo ANA Sal Mo Olfactory tubercle AA Saline Mo ANA Saline Mo
Dopamine
DOPAC
HVA
3-MT
5-HT
5-HIAA
14700 6 310 15800 6 1500
890 6 30 1130 6 120
700 6 20 1040 6 90†
13.8 6 0.7 16.8 6 1.8
590 6 30 600 6 50
470 6 20 540 6 40
11000 6 600 11250 6 750
720 6 50 900 6 40
610 6 50 850 6 40*
15.3 6 2.1 15.2 6 1.9
390 6 50 480 6 50
400 6 20 450 6 20
9920 6 400 9600 6 590
900 6 140 1270 6 190
650 6 40 990 6 90†
5.7 6 0.3 8.0 6 1.3
1270 6 80 1140 6 40
600 6 40 620 6 40
9530 6 380 7870 6 460*
880 6 140 1010 6 140
640 6 50 850 6 70
7.7 6 0.4 8.6 6 1.7
1060 6 70 990 6 50
430 6 20 480 6 30
6600 6 170 6560 6 400
670 6 40 870 6 60*
430 6 20 670 6 50†
6.3 6 0.4 7.7 6 0.4
1710 6 90 1760 6 60
390 6 30 470 6 10*
5670 6 340 5830 6 280
630 6 50 860 6 50*
420 6 30 650 6 50†
6.4 6 0.2 7.9 6 0.6
1460 6 70 1480 6 70
360 6 10 340 6 20
*p , 0.05, †p , 0.01 in comparison with corresponding saline-treated group (Tukey compromise test, n 5 7–8).
DISCUSSION
Decreased Basal Dopamine Release in Alcohol-Preferring Rats We found that 3-MT concentration was lower both in the caudate-putamen and nucleus accumbens of the alcohol-preferring AA rats than in the alcohol-avoiding ANA rats. 3-MT is formed from dopamine outside of the dopaminergic neurons by catechol-O-methyltransferase (COMT), implying that its concentration in tissue reflects dopamine release (61). Therefore, our finding indicates that the basal dopamine release in the caudate-putamen and nucleus accumbens is lower in the AA rats than in the ANA rats. The basal 3-MT concentration in the brains of the AA and ANA rats has been assessed only once before, and in that work a similar tendency for a line difference in 3-MT concentration was found (20). In addition to lowered dopamine release, smaller brain 3-MT concentration of the AA rats could be due to the lower activity of COMT in the AA rats than ANA rats. This is unlikely, however, since there is no difference in the brain COMT activity between AA and ANA rats (46), and furthermore, the concentration of HVA, which is also a product of COMT (61), does not differ between the AA and ANA rats (35). Extracellular dopamine concentrations in the nucleus accumbens of the AA and ANA rats have also been estimated with in vivo microdialysis but no significant differences were found (30,43). This is not surprising, since there is a large animal to animal variation in the basal dopamine concentrations in the dialysates, which makes it difficult to detect relatively small differ-
ences in extracellular dopamine concentrations with this method. Interestingly, it has been proposed that a deficient dopaminergic signaling may be a factor predisposing an individual to high alcohol consumption (12,39,54,59). Alcoholics and alcohol-preferring experimental animals might try to compensate this deficit by alcohol drinking. In line with this theory, pharmacological activation of cerebral dopamine receptors decrease alcohol drinking in some rodent lines (9,12,49). Therefore, it would be interesting to study whether those agents modulate alcohol drinking in the AA rats as well. Enhanced Morphine-Induced Dopamine Release in the Caudate-Putamen of Alcohol-Preferring Rats The main goal of this work was to examine whether the greater stimulation of locomotor activity induced by morphine in the AA rats than in the ANA rats (21) is associated with a greater dopamine release in brains of the AA rats. There was no difference in the morphine-induced increase of dopamine release in the nucleus accumbens and olfactory tubercle between the rat lines, suggesting that differential locomotor stimulation by acute morphine does not depend on mesolimbic dopamine release. In contrast, the nigrostriatal pathway was more activated by morphine in the AA rats than ANA rats suggesting that large morphine-induced locomotor stimulation in the AA rats is, at least partially, due to strong activation of dopamine release in the caudate-putamen. This is inconsistent with the idea that locomotor activity is regulated by
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dopamine in the ventral striatum, i.e. nucleus accumbens and olfactory tubercle rather than in the caudate-putamen (29). In keeping with the differential roles of these two dopaminergic pathways in the regulation of motor activity, local m-agonists induce locomotor stimulation in the VTA but not in the substantia nigra, in which they usually induce stereotyped behavior (26,27,41). On the other hand, there is some evidence that m-opioids may induce locomotor activity also in the substantia nigra via a specific subpopulation of m-receptors, when a portion of the opioid receptors is blocked with small dose of naloxone (41). Furthermore, we recently found that density of m-receptors is higher in the substantia nigra pars reticulata of the AA than ANA rats (51). Therefore, it is still possible that nigrostriatal dopamine pathway plays an important role in high reactivity of the AA rats to morphine. However, it is clear that in addition to substantia nigra and VTA, there are several other brain areas, where m-opioids induce locomotor stimulation (6,28,31). When infused locally into the nucleus accumbens, m-opioids cause locomotor stimulation that can not be blocked by dopamine receptor antagonists (28,45). Therefore, it is possible that the differential morphine-induced locomotor stimulation between the rat lines is partially due to a difference in nondopamine dependent mechanisms in the nucleus accumbens. Our finding that the nigrostriatal pathway of the alcoholpreferring rats is more sensitive to opioidergic stimulation than that of the alcohol-avoiding rats suggests its involvement in the regulation of alcohol drinking. This is somewhat surprising as motivational effects of drugs of abuse have been usually linked to dopamine mechanisms in the ventral striatum, but not to those in the caudate-putamen (33,58). Consistent with this, we recently found that dopamine release (tissue 3-MT concentration) was increased selectively in the nucleus accumbens and olfactory tubercle of the AA rats after a bout of voluntary alcohol drinking in a limited access paradigm (19). On the other hand, dopamine release in the caudateputamen was similarly increased in rats drinking alcohol and in those given plain water instead alcohol in the alcohol drinking session. Since both of these groups had similar alcohol drinking histories before final experimental session, this finding suggest that motor processes involved in drinking or environmental cues associated with drinking session were able to activate dopaminergic neurons projecting to the caudate-putamen. Dopaminergic system of the caudate-putamen has been suggested to be involved in learning of stimulus-response associations and coordination of complex instrumental responses induced by conditioned stimuli (50,55). Furthermore, there is some evidence about involvement of nigrostriatal dopamine system in food reinforcement (8). It is possible that these functions of dopamine in the caudate-putamen and their opioidergic modulation contribute to, e.g., rapid learning of alcohol drinking behavior in the AA rats. Therefore, it is clear that the role of the nigrostriatal pathway in the differential alcohol preference in the AA and ANA rats needs to be evaluated in further studies.
Role of 5-HT in the Enhanced Morphine Effects in the AA Rats The present findings showed that morphine at low doses elevated 5-HT metabolism both in the ventral and dorsal striatum of the AA rats but not in those of the ANA rats, suggesting that the sensitivity of 5-HT mechanisms to opioidergic modulation differs between these rat lines. The 5-HT system have been implicated in the regulation of motor behavior as well as in the regulation of alcohol drinking (13,15,38,40,42,57). 5-HT receptor ligands also modulate the function of dopaminergic neurons (7,42), suggesting that 5-HT may produce its behavioral effects, at least partly, by accelerating dopamine release. Locomotor stimulation is induced by agonists for 5-HT3 receptors, probably via acceleration of dopamine release (15) and by 5HT1B agonists via dopamine-independent mechanisms (13). Therefore, it is possible that these receptors are involved in the differential morphine-induced locomotor and neurochemical response between the AA and ANA rats. Reduced 5-HT activity in brain is believed to be important in the certain forms of excessive alcohol consumption. Low 5-HT levels and receptor densities have been found in the brains of alcohol-preferring P and HAD rats (40) and low levels of 5-HIAA in the cerebrospinal fluid of early-onset, male alcoholics (38,57). AA and ANA rats, however, appear to be different from the above examples, since levels of 5-HT are higher in the brains of the alcohol-preferring AA rats than in the alcohol-avoiding ANAs (5,35), as found also in the present study. Furthermore, there is no differences in the ligand binding to 5-HT1, 5-HT2 and 5-HT3 receptors between the brains of the AA and ANA rats (34). The present work, however, provides evidence that the sensitivity of 5-HTergic neurons to opioids differs between the AA and ANA rats. Therefore, the involvement of the 5-HT system as a factor contributing to the differential alcohol preference of the AA and ANA rats can not be entirely excluded. In summary, we found that the basal dopamine release is low both in the dorsal and ventral striatum of the alcohol-preferring AA rats, a trait possibly making this rat line prone to high alcohol intake. The nigrostriatal but not the mesolimbic dopaminergic pathway of the AA rats seems to be more activated by low morphine doses in the AA rats than in the ANA rats. Furthermore, the metabolism of 5-HT was significantly enhanced by morphine in the brains of the AA rats but not in those of the ANA rats. As alcohol is believed to activate brain opioidergic mechanisms, we suggest that the high sensitivity of the brain dopaminergic and/or 5-HTergic mechanisms of the AA rats to opioid-induced stimulation as compared with the ANA rats may play a role in the differential alcohol preference of these rat lines.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Finnish Cultural Foundation to A.H.
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HONKANEN ET AL. 55. White, N. M.: Mnemonic functions of the basal ganglia. Curr. Opin. Neurobiol. 7:164–169; 1997. 56. Widdowson, P. S.; Holman, R. B.: Ethanol-induced increase of endogenous dopamine release may involve endogenous opiates. J. Neurochem. 59:157–163; 1992. 57. Virkkunen, M.; Linnoila, M.: Serotonin in early-onset alcoholism. In: Galanter, M., ed., Alcoholism and Violence, Recent Developments in Alcoholism, vol. 13. New York: Pleunum Press; 1997: 173–189. 58. Wise, R. A.; Bozarth, M. A.: A psychomotor stimulant theory of addiction. Psychol. Rev. 94:469–492; 1987. 59. Volkov, N. D.; Wang, G.-J.; Fowler, J. S.; Logan, J.; Hitzemann, R.; Ding, Y.-S.; Pappas, N.; Shea, C.; Piscani, K.: Decrease in dopamine receptors but not in dopamine transporters in alcoholics. Alcohol. Clin. Exp. Res. 20:1594–1598; 1996. 60. Volpicelli, J. R.; Volpicelli, L. A.; O’Brien, C. P.: Medical management of alcohol dependence: clinical use and limitations of naltrexone treatment. Alcohol Alcohol. 30:789–798; 1995. 61. Wood, P. L.; Altar, C. A.: Dopamine release in vivo from nigrostriatal, mesolimbic, and mesocortical neurons: Utility of 3-methoxytyramine measurements. Pharmacol. Rev. 40:163–187; 1988.
PII S0741-8329(98)00060-3
Effects of Morphine on Metabolism of Dopamine and Serotonin in Brains of Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats AAPO HONKANEN,*‡ PETRI HYYTIÄ,† ESA R. KORPI†‡ AND LIISA AHTEE* *Department of Pharmacy, Division of Pharmacology and Toxicology,University of Helsinki, POB 56, FIN-00014, University of Helsinki, Finland; †Department of Mental Health and Alcohol Research, National Public Health Institute, POB 719, FIN-00101, Helsinki, Finland; ‡Department of Pharmacology and Clinical Pharmacology, University of Turku, FIN-20520, Turku, Finland Received 22 April 1998; Accepted 6 October 1998 HONKANEN, A., P. HYYTIÄ, E. R. KORPI AND L. AHTEE. Effects of morphine on metabolism of dopamine and serotonin in brains of alcohol-preferring AA and alcohol-avoiding ANA rats. ALCOHOL 18(1) 3–10, 1999.—Morphine induces a larger locomotor stimulation in the alcohol-preferring AA rats than in the alcohol-avoiding ANA rats. We have now studied the acute effects of morphine (1 and 3 mg/kg) on metabolism of dopamine and serotonin (5-HT) in the dorsal and ventral striatum of the AA and ANA rats. The basal level of dopamine release, as reflected by the concentration of dopamine metabolite 3-methoxytyramine (3-MT), was lower in the caudate-putamen and nucleus accumbens of the AA rats than in the ANA rats. In the caudate-putamen, morphine increased dopamine metabolism and release more in the AA than in the ANA rats. In the nucleus accumbens and olfactory tubercle, the effects of morphine on dopamine metabolism and release did not differ between the rat lines. Morphine elevated the metabolism of 5-HT in the caudate-putamen and nucleus accumbens of the AA but not in those of the ANA rats. The results suggest that the larger morphine-induced psychomotor stimulation of the AA rats in comparison with the ANA rats is associated with the larger effect of morphine on dopamine metabolism in the caudate-putamen and 5-HT metabolism in the caudate-putamen and nucleus accumbens. Furthermore, low basal dopamine release may play a role in the high alcohol-preference of AA rats. © 1999 Elsevier Science Inc. All rights reserved. Alcohol preference Nucleus accumbens
Alcohol avoidance Caudate-Putamen
Dopamine Morphine 3-Methoxytyramine Olfactory tubercle Selected rat lines
tides (14) and dopamine (24) and also change acutely and/or chronically opioid (10,53) and dopamine (23) receptors in brain. Furthermore, blockade of opioid receptors antagonizes the stimulatory effects of alcohol on dopamine metabolism and release in experimental animals (1,48,56). These findings suggest that the effects of alcohol on dopaminergic mechanisms could be mediated by endogenous opioids; i.e., the alcohol-induced release of endogenous opioids could stimulate dopamine release similarly as exogenous opioids do. The in-
VARIOUS drugs of abuse are believed to cause their reinforcing effects, at least partly, by stimulating dopamine release in the nucleus accumbens through various primary mechanisms (33,58). For instance, m-opioid receptor agonists, such as heroin and morphine, increase activity of dopamine neurons by disinhibiting dopaminergic cells in the VTA (16,25), which leads to increased dopamine release in the nucleus accumbens (27,37). Alcohol is believed to increase the release of opioid pep-
Requests for reprints should be addressed to Aapo Honkanen, Department of Pharmacology and Clinical Pharmacology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Tel: 1358-2-333-7678; Fax: 1358-2-333-7000; E-mail: [email protected]
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volvement of brain opioidergic system in the regulation of alcohol drinking is supported by findings that blockade of opioid receptors decreases alcohol drinking in experimental animals and humans (18,60). Acceleration of dopamine release in the nucleus accumbens results also in psychomotor stimulation, which, for example in rats and mice, is seen as increased locomotor activity (36,58). In a recent study, we found that morphine, a relatively selective m-opioid receptor agonist, induces larger locomotor stimulation in alcohol-preferring AA (Alko, Alcohol) rats than in alcohol-avoiding ANA (Alko, Nonalcohol) or nonselected Wistar rats (21). Based on the theory of homology between psychomotor stimulation and reinforcement (58), this finding suggests that alcohol-preferring rats are hypersensitive to the reinforcing effects of opioids. Therefore, a possible mechanism underlying the high alcohol-preference of AA rats could be a strong alcohol-induced activation of opioid systems, which could, in turn, cause enhanced dopamine release in these rats. Alternatively, m-opioid receptor agonists may induce both locomotor stimulation and reinforcement via dopamine-independent mechanisms, e.g., by acting on nondopaminergic cells in the nucleus accumbens (11). The aim of the present study was to investigate whether the effects of morphine on metabolism and release of dopamine in the dorsal (caudate-putamen) and ventral striatum (nucleus accumbens and olfactory tubercle) differ in the AA and ANA rats. Morphine-induced changes in the caudateputamen were assessed because it is possible that motor effects of opioids are partially mediated by this brain region (2). The olfactory tubercle was included in the study because, in addition to the nucleus accumbens, dopaminergic mechanism of this brain regions may be involved in regulation of alcohol drinking (19,47). Previously, it has been shown that concentrations of serotonin (5-hydroxytryptamine, 5-HT) are larger in the brains of the AA rats than in the ANA rats (5) suggesting involvement of this neurotransmitter in the differential alcohol preference of these animals. Like dopamine, 5-HT mechanisms are regulated by opioids (32,52) and they are involved in the regulation of alcohol consumption both in experimental animals (40) and humans (38,57). Furthermore, 5-HT regulates dopaminergic neurons (7,42) and locomotor activity (13,15,42). Therefore, morphine effects on metabolism of 5-HT were also assessed in the AA and ANA rats. METHOD
Animals Three-months-old AA and ANA rats from generations F69 and F72 (Department of Mental Health and Alcohol Research, National Public Health Institute) were used. The animals were housed in stainless-steel wire-mesh cages in groups of 4–5 animals per cage. The animals were maintained on a 12-h dark/light cycle (lights on from 600 to 1800 h), and ambient temperature of 20 6 28C and the relative air humidity of 50 6 5%. R3 rat pellet food (Ewos AB, Södertälje, Sweden) and tap water were available ad libitum. Written permissions for all experiments were obtained from the experimental animal committee of the National Public Health Institute. Experimental Procedures In experiment 1, the animals (n 5 8–11 per group) were given saline (1 ml/kg, SC) or morphine hydrochloride (Ph. Eur) at the doses of 1 or 3 mg/kg (as base in saline, SC). The
animals were killed 60 min after injections with head-focused microwave irradiation by using the NJE 2603-10 kW microwave applicator (New Japan Radio Inc., Japan) set at output power of 7 kW and irradiation time of 1.4 s. Experiment 2 was carried out in order to get more information on the time course of the effects of morphine on dopamine metabolism. The animals were killed 120 min after drug administration, and due to limited availability of the AA and ANA rats, only one morphine dose, 1 mg/kg, was used. During the time period between experiments 1 and 2, we found that voluntary alcohol drinking increases dopamine release not only in the the nucleus accumbens, but also in the olfactory tubercle of the AA rats (19). Therefore, the latter brain region was included in experiment 2 as well. After killing, the animals were decapitated and their heads were allowed to cool in crushed ice for 60 s. Then the brains of the animals were removed from the skulls, placed in a zinc brain mold (RBM-4000C, ASI Instruments, USA) cooled on ice, and sectioned coronally with razor blades at 2.7, 20.3 and 24.3 mm from bregma (44). The caudate-putamen and the nucleus accumbens were dissected from the second slice as described earlier (20) and the tissue including olfactory tubercle in the second slice located ventral to the nucleus accumbens was also dissected. The tissues were immediately frozen on dry ice and stored at 2808C before assayed for dopamine, 5-HT and their metabolites. Estimation of Brain Monoamines and Their Metabolites Samples were homogenized in 1 ml of 0.2 M HClO4 after which 25 ml KOH/HCOOH buffer was added to the homogenates to adjust the pH to 2.4. Samples were centrifuged first at 5,500 3 g for 45 min and then at 28,000 3 g for 20 min. The supernatants were purified using the method described earlier (17). In brief, a 950 ml sample of supernatant was pipetted onto Sephadex G-10 columns and washed with 1.8 ml of 0.01 M HCl. Dopamine and 3-MT were collected with 1.7 ml 0.01 M HCl and 1.0 ml 0.02 M NH3. The acidic metabolites HVA and DOPAC were collected by washing the columns with 1.0 ml of 0.02 M NH3 and 1.0 ml of 0.01 M KOH. Twenty ml of 2.6 mM sodium pyrosulfite and 5.7 mM ascorbic acid were added into the tubes containing dopamine/3-MT and DOPAC/ HVA, respectively. The samples were assayed for the concentration of dopamine and its metabolites by using HPLC with electrochemical detection as described earlier (22). Statistics Differences in the basal concentrations of monoamines and their metabolites between the rat lines were tested with Student’s t-test with Bonferroni adjustment. Drug effects, rat line differences and interaction between rat line and drug effect were evaluated first by using two-way analysis of variance (2-way ANOVA) followed by the Tukey compromise posthoc test. Since the two experiments conducted differed in design, the data from these experiments were tested separately with ANOVA. RESULTS
Basal Concentrations of Dopamine, 5-HT and Their Metabolites In order to compare the basal concentrations of monoamines and their metabolites between the rat strains, the data from the control groups of the two experiments were combined and are presented in Table 1. Dopamine concentrations were not
MORPHINE EFFECTS IN AA AND ANA RATS
5 TABLE 1
THE MEAN BASAL CONCENTRATIONS (6 SE) OF DOPAMINE AND ITS METABOLITES [3,4-DIHYDROXYPHENYL ACETIC ACID (DOPAC), HOMOVANILLIC ACID (HVA) AND 3-METHOXYTYRAMINE (3-MT)] AND 5-HYDROXYTRYPTAMINE (5-HT) AND ITS METABOLITE 5-HYDROXYINDOLEACETIC ACID (5-HIAA) IN DIFFERENT BRAIN AREAS OF ALCOHOL-PREFERRING AA AND ALCOHOL-AVOIDING ANA RATS Concentration (ng/g) Dopamine
Caudate putamen AA ANA Nucleus Accumbens AA ANA Olfactory tubercle AA ANA
DOPAC
HVA
3-MT
5-HT
5-HIAA
620 6 30 450 6 25†
470 6 10 400 6 15
14700 6 310 11000 6 640†
920 6 40 900 6 70
750 6 30 720 6 40
12.4 6 0.5 15.0 6 0.8*
9900 6 410 9500 6 380
960 6 70 860 6 100
640 6 30 630 6 25
5.3 6 0.2 6.5 6 0.4†
1390 6 60 1210 6 70
610 6 20 540 6 20
6600 6 170 5700 6 340
670 6 40 630 6 50
430 6 20 420 6 30
6.2 6 0.4 6.5 6 0.2
1700 6 90 1460 6 70
400 6 30 360 6 10
*p , 0.05, †p , 0.01 in comparison with AA rats (Student’s t-test with Bonferroni adjustment, n 5 8–19).
estimated from samples of the first experiment, so the data shown is from the experiment 2 only, as well as the data from the olfactory tubercle, which was not studied in the first experiment. In the caudate-putamen, the concentration of 3-MT was smaller in the AA than in the ANA rats, but, as previously reported (3,4,5), the concentrations of dopamine, 5-HT and 5-HIAA were higher in AA rats than in the ANA rats. The concentrations of the DOPAC and HVA did not differ in this brain region between the rat lines. In the nucleus accumbens, the 3-MT concentration was again smaller in the AA rats than in the ANA rats, while other meausures did not differ significantly between the rat lines. In the olfactory tubercle, concentration of 5-HT tended to be higher in the AA rats than ANA rats, but the diference did not reach statistical significance. Morphine Effects 60 min After Treatment (Experiment 1) In the caudate-putamen, 2-way ANOVA showed a rat line, F(1, 56) 5 8.65, p , 0.01 and treatment effects, F (2, 56) 5 12.8, P , 0.001; but no rat x treatment interaction F (2, 56) 5 0.86, p 5 0.43 for 3-MT. Posthoc comparison, however, revealed that 3-MT levels were significantly elevated by both morphine doses in the AA rats but not in the ANAs (Fig. 1). For DOPAC, there were significant rat line, F (1, 56) 5 5.78, p , 0.05 and treatment effects, F (2, 56) 5 50.4, p , 0.001 as well as significant rat line x treatment interaction F (2, 56) 5 3.8, p , 0.05, which was apparently due to larger elevation of DOPAC levels in the AA rats than in the ANAs. Similarly, ANOVA showed a rat line, F (1, 56) 5 8.43, p , 0.01 and treatment effects, F (2, 56) 5 44.8, p , 0.001, and rat line x treatment interaction, F (2, 56) 5 3.29, p , 0.05 for HVA. Morphine-induced elevation of HVA concentrations was greater in the AA rats than in the ANA rats. Morphine did not alter 5-HT concentration in the caudateputamen: treatment effect, F (2, 56) 5 1.47, p 5 0.24, but there was a significant rat line effect, F (1, 56) 5 28.9, p , 0.001 and no interaction, F (2, 56) 5 0.83, p 5 0.44. For 5-HIAA, rat line and treatment effects were highly significant, F (1, 56) 5 43.6. p , 0.001 and F (2, 56) 5 10.7. p , 0.001, respectively, but no interaction between them were found, F (2, 56) 5 1.62, p 5 0.21. Posthoc test indicated that 5-HIAA levels were significantly elevated by morphine in the AA rats only.
In the nucleus accumbens, ANOVA showed significant rat line, F (1, 55) 5 6.74 p , 0.05, and treatment effects, F (2, 56) 5 23.7, p , 0.001, for 3-MT, but no rat line x treatment interaction, F (2, 56) 5 2.93, p 5 0.06. Significant treatment effects were found for DOPAC, F (2, 55) 5 15.6, p , 0.001, and HVA, F (2, 55) 5 81.4, p , 0.001, but no rat line effects, F (1, 55) 5 0.008, p 5 0.93 (for DOPAC) and F (1, 55) 5 1.21, p 5 0.28 (for HVA), or interactions were found F (2, 56) 5 1.45, p 5 0.25 (for DOPAC) and F (1, 55) 5 0.27, p 5 0.77 (for HVA). For 5-HT, ANOVA showed a significant rat line effect, F (1, 54) 5 9.87, p , 0.01, but no treatment effect, F (2, 54) 5 1.12, p 5 0.33, or interaction, F (2, 54) 5 2.55, p 5 0.088. For 5-HIAA, ANOVA showed highly significant rat line effect, F (1, 55) 5 36.2, p , 0.001, and treatment effect, F (2, 55) 5 8.78, p , 0.001, but no interaction between them, F (2, 55) 5 1.95, p 5 0.15. Posthoc test, however, showed that 5-HIAA levels were elevated in the AA rats but not in the ANAs. Morphine Effects 120 min After Treatment (Experiment 2) In the caudate-putamen, two h after the morphine treatment (1 mg/kg), significant treatment effects were found for DOPAC, F (1, 28) 5 8.59, p , 0.01; HVA, F (1, 28) 5 27.1, p , 0.001, and 5-HIAA, F (1, 28) 5 5.72, p , 0.05, but not for dopamine, F (1, 28) 5 0.56, p 5 0.46 and 3-MT, F (1, 25) 5 0.67, p 5 0.42, and no rat line x treatment interactions were found (Table 2). Significant rat line effects were found for dopamine, F (1,28) 5 20.0, p , 0.001; DOPAC, F (1,28) 5 8.59, p , 0.01; HVA, F (1,28) 5 6.14, p , 0.05; 5-HT, F (1,28) 5 12.9, p , 0.01 and 5-HIAA, F (1,28) 5 10.7, p , 0.01, but not for 3-MT. In posthoc comparisons only HVA concentrations were elevated in both rat lines. In the nucleus accumbens, the concentrations of dopamine, F (1, 28) 5 4.51, p , 0.05 and HVA, F (1, 28) 5 18.1, p , 0.001 were altered by morphine treament, but not those of 3-MT, F (1, 28) 5 2.05, P 5 0.16; DOPAC, F (1, 28) 5 2.56, p , 0.12 and 5-HIAA, F (1, 28) 5 0.22, p , 0.64 (Table 2). ANOVA showed a significant rat line effects for dopamine, F (1, 28) 5 5.10, p , 0.05; 5-HT, F (1, 28) 5 7.78, p , 0.01, and 5-HIAA, F (1,28) 5 10.1, p , 0.01, but not for other measures. Posthoc test showed that dopamine was significantly decreased by morphine in the ANA rats but not in the AA rats, and HVA was significantly increased in the AA rats only.
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FIG. 1. Effects of morphine (1 or 3 mg/kg, s.c.) or saline (SAL) on the dopamine metabolites, 3-methoxytyramine (3-MT), homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid DOPAC and on the 5-HT metabolite 5-hydroxyindolacetic acid (5-HIAA) in the caudate-putamen and in the nucleus accumbens of the AA and ANA (experiment 1). Rats were given morphine or 0.9 % NaCl (SAL) and killed 60 min later with head-focused microwave irradiation. *p , 0.05 and **p , 0.01, in comparison with respective SAL-treated control group (Tukey compromise test).
In the olfactory tubercle, ANOVA showed a significant treatment effects for 3-MT, F (1, 27) 5 10.9, p , 0.05; DOPAC, F (1, 27) 5 16.7, p , 0.01; HVA, F (1, 27) 5 40.5, p , 0.001, but not for dopamine, F (1, 27) 5 0.04, p 5 0.85; 5-HT, F (1, 27) 5 0.28, p , 0.60 or 5-HIAA, F (1, 27) 5 1.92, p , 0.18 (Table 2). A significant rat line x treatment interaction
was found only for 5-HIAA, F (1, 27) 5 5.28, p , 0.05. ANOVA showed a significant rat line effects for dopamine, F (1,27) 5 7.64, p , 0.05; for 5-HT, F (1,27) 5 13.8, p , 0.001 and for 5-HIAA, F (1,27) 5 14.5, p , 0.001. Posthoc test showed that DOPAC and HVA concentrations were elevated in both rat lines, but 5-HIAA only in the AA rats.
MORPHINE EFFECTS IN AA AND ANA RATS
7 TABLE 2
EFFECTS OF MORPHINE (MO, 1 mg/kg, SC) ON THE CONCENTRATIONS DOPAMINE AND ITS METABOLITES [3,4-DIHYDROXYPHENYL ACETIC ACID (DOPAC), HOMOVANILLIC ACID (HVA) AND 3-METHOXYTYRAMINE (3-MT)] AND 5-HYDROXYTRYPTAMINE (5-HT) AND ITS METABOLITE 5-HYDROXYINDOLACETIC ACID (5-HIAA) IN DIFFERENT BRAIN AREAS OF ALCOHOL-PREFERRING AA AND ALCOHOL-AVOIDING ANA RATS 120 MIN AFTER INJECTIONS (EXPERIMENT 2) Concentration (ng/g)
Caudate-Putamen AA Saline Mo ANA Sal Mo Nucleus accumbens AA Saline Mo ANA Sal Mo Olfactory tubercle AA Saline Mo ANA Saline Mo
Dopamine
DOPAC
HVA
3-MT
5-HT
5-HIAA
14700 6 310 15800 6 1500
890 6 30 1130 6 120
700 6 20 1040 6 90†
13.8 6 0.7 16.8 6 1.8
590 6 30 600 6 50
470 6 20 540 6 40
11000 6 600 11250 6 750
720 6 50 900 6 40
610 6 50 850 6 40*
15.3 6 2.1 15.2 6 1.9
390 6 50 480 6 50
400 6 20 450 6 20
9920 6 400 9600 6 590
900 6 140 1270 6 190
650 6 40 990 6 90†
5.7 6 0.3 8.0 6 1.3
1270 6 80 1140 6 40
600 6 40 620 6 40
9530 6 380 7870 6 460*
880 6 140 1010 6 140
640 6 50 850 6 70
7.7 6 0.4 8.6 6 1.7
1060 6 70 990 6 50
430 6 20 480 6 30
6600 6 170 6560 6 400
670 6 40 870 6 60*
430 6 20 670 6 50†
6.3 6 0.4 7.7 6 0.4
1710 6 90 1760 6 60
390 6 30 470 6 10*
5670 6 340 5830 6 280
630 6 50 860 6 50*
420 6 30 650 6 50†
6.4 6 0.2 7.9 6 0.6
1460 6 70 1480 6 70
360 6 10 340 6 20
*p , 0.05, †p , 0.01 in comparison with corresponding saline-treated group (Tukey compromise test, n 5 7–8).
DISCUSSION
Decreased Basal Dopamine Release in Alcohol-Preferring Rats We found that 3-MT concentration was lower both in the caudate-putamen and nucleus accumbens of the alcohol-preferring AA rats than in the alcohol-avoiding ANA rats. 3-MT is formed from dopamine outside of the dopaminergic neurons by catechol-O-methyltransferase (COMT), implying that its concentration in tissue reflects dopamine release (61). Therefore, our finding indicates that the basal dopamine release in the caudate-putamen and nucleus accumbens is lower in the AA rats than in the ANA rats. The basal 3-MT concentration in the brains of the AA and ANA rats has been assessed only once before, and in that work a similar tendency for a line difference in 3-MT concentration was found (20). In addition to lowered dopamine release, smaller brain 3-MT concentration of the AA rats could be due to the lower activity of COMT in the AA rats than ANA rats. This is unlikely, however, since there is no difference in the brain COMT activity between AA and ANA rats (46), and furthermore, the concentration of HVA, which is also a product of COMT (61), does not differ between the AA and ANA rats (35). Extracellular dopamine concentrations in the nucleus accumbens of the AA and ANA rats have also been estimated with in vivo microdialysis but no significant differences were found (30,43). This is not surprising, since there is a large animal to animal variation in the basal dopamine concentrations in the dialysates, which makes it difficult to detect relatively small differ-
ences in extracellular dopamine concentrations with this method. Interestingly, it has been proposed that a deficient dopaminergic signaling may be a factor predisposing an individual to high alcohol consumption (12,39,54,59). Alcoholics and alcohol-preferring experimental animals might try to compensate this deficit by alcohol drinking. In line with this theory, pharmacological activation of cerebral dopamine receptors decrease alcohol drinking in some rodent lines (9,12,49). Therefore, it would be interesting to study whether those agents modulate alcohol drinking in the AA rats as well. Enhanced Morphine-Induced Dopamine Release in the Caudate-Putamen of Alcohol-Preferring Rats The main goal of this work was to examine whether the greater stimulation of locomotor activity induced by morphine in the AA rats than in the ANA rats (21) is associated with a greater dopamine release in brains of the AA rats. There was no difference in the morphine-induced increase of dopamine release in the nucleus accumbens and olfactory tubercle between the rat lines, suggesting that differential locomotor stimulation by acute morphine does not depend on mesolimbic dopamine release. In contrast, the nigrostriatal pathway was more activated by morphine in the AA rats than ANA rats suggesting that large morphine-induced locomotor stimulation in the AA rats is, at least partially, due to strong activation of dopamine release in the caudate-putamen. This is inconsistent with the idea that locomotor activity is regulated by
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dopamine in the ventral striatum, i.e. nucleus accumbens and olfactory tubercle rather than in the caudate-putamen (29). In keeping with the differential roles of these two dopaminergic pathways in the regulation of motor activity, local m-agonists induce locomotor stimulation in the VTA but not in the substantia nigra, in which they usually induce stereotyped behavior (26,27,41). On the other hand, there is some evidence that m-opioids may induce locomotor activity also in the substantia nigra via a specific subpopulation of m-receptors, when a portion of the opioid receptors is blocked with small dose of naloxone (41). Furthermore, we recently found that density of m-receptors is higher in the substantia nigra pars reticulata of the AA than ANA rats (51). Therefore, it is still possible that nigrostriatal dopamine pathway plays an important role in high reactivity of the AA rats to morphine. However, it is clear that in addition to substantia nigra and VTA, there are several other brain areas, where m-opioids induce locomotor stimulation (6,28,31). When infused locally into the nucleus accumbens, m-opioids cause locomotor stimulation that can not be blocked by dopamine receptor antagonists (28,45). Therefore, it is possible that the differential morphine-induced locomotor stimulation between the rat lines is partially due to a difference in nondopamine dependent mechanisms in the nucleus accumbens. Our finding that the nigrostriatal pathway of the alcoholpreferring rats is more sensitive to opioidergic stimulation than that of the alcohol-avoiding rats suggests its involvement in the regulation of alcohol drinking. This is somewhat surprising as motivational effects of drugs of abuse have been usually linked to dopamine mechanisms in the ventral striatum, but not to those in the caudate-putamen (33,58). Consistent with this, we recently found that dopamine release (tissue 3-MT concentration) was increased selectively in the nucleus accumbens and olfactory tubercle of the AA rats after a bout of voluntary alcohol drinking in a limited access paradigm (19). On the other hand, dopamine release in the caudateputamen was similarly increased in rats drinking alcohol and in those given plain water instead alcohol in the alcohol drinking session. Since both of these groups had similar alcohol drinking histories before final experimental session, this finding suggest that motor processes involved in drinking or environmental cues associated with drinking session were able to activate dopaminergic neurons projecting to the caudate-putamen. Dopaminergic system of the caudate-putamen has been suggested to be involved in learning of stimulus-response associations and coordination of complex instrumental responses induced by conditioned stimuli (50,55). Furthermore, there is some evidence about involvement of nigrostriatal dopamine system in food reinforcement (8). It is possible that these functions of dopamine in the caudate-putamen and their opioidergic modulation contribute to, e.g., rapid learning of alcohol drinking behavior in the AA rats. Therefore, it is clear that the role of the nigrostriatal pathway in the differential alcohol preference in the AA and ANA rats needs to be evaluated in further studies.
Role of 5-HT in the Enhanced Morphine Effects in the AA Rats The present findings showed that morphine at low doses elevated 5-HT metabolism both in the ventral and dorsal striatum of the AA rats but not in those of the ANA rats, suggesting that the sensitivity of 5-HT mechanisms to opioidergic modulation differs between these rat lines. The 5-HT system have been implicated in the regulation of motor behavior as well as in the regulation of alcohol drinking (13,15,38,40,42,57). 5-HT receptor ligands also modulate the function of dopaminergic neurons (7,42), suggesting that 5-HT may produce its behavioral effects, at least partly, by accelerating dopamine release. Locomotor stimulation is induced by agonists for 5-HT3 receptors, probably via acceleration of dopamine release (15) and by 5HT1B agonists via dopamine-independent mechanisms (13). Therefore, it is possible that these receptors are involved in the differential morphine-induced locomotor and neurochemical response between the AA and ANA rats. Reduced 5-HT activity in brain is believed to be important in the certain forms of excessive alcohol consumption. Low 5-HT levels and receptor densities have been found in the brains of alcohol-preferring P and HAD rats (40) and low levels of 5-HIAA in the cerebrospinal fluid of early-onset, male alcoholics (38,57). AA and ANA rats, however, appear to be different from the above examples, since levels of 5-HT are higher in the brains of the alcohol-preferring AA rats than in the alcohol-avoiding ANAs (5,35), as found also in the present study. Furthermore, there is no differences in the ligand binding to 5-HT1, 5-HT2 and 5-HT3 receptors between the brains of the AA and ANA rats (34). The present work, however, provides evidence that the sensitivity of 5-HTergic neurons to opioids differs between the AA and ANA rats. Therefore, the involvement of the 5-HT system as a factor contributing to the differential alcohol preference of the AA and ANA rats can not be entirely excluded. In summary, we found that the basal dopamine release is low both in the dorsal and ventral striatum of the alcohol-preferring AA rats, a trait possibly making this rat line prone to high alcohol intake. The nigrostriatal but not the mesolimbic dopaminergic pathway of the AA rats seems to be more activated by low morphine doses in the AA rats than in the ANA rats. Furthermore, the metabolism of 5-HT was significantly enhanced by morphine in the brains of the AA rats but not in those of the ANA rats. As alcohol is believed to activate brain opioidergic mechanisms, we suggest that the high sensitivity of the brain dopaminergic and/or 5-HTergic mechanisms of the AA rats to opioid-induced stimulation as compared with the ANA rats may play a role in the differential alcohol preference of these rat lines.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Finnish Cultural Foundation to A.H.
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