Alterations in dopamine metabolism by intraperitoneal ethanol in rats selected for high and low ethanol preference: A 3-methoxytyramine study

Alcohol, Vol. 11, No. 4, pp. 323-328, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0741-8329/94 $6.00 + .00

Pergamon 0741-8329(94)E0013-3

Alterations in Dopamine Metabolism by Intraperitoneal Ethanol in Rats Selected for High and Low Ethanol Preference: A 3-Methoxytyramine Study A. HONKANEN,*

S. J. C H R A P U S T A , t

F. K A R O U M t

AND

E. R. K O R P I *l

*Biomedical Research Center, A l k o Ltd, P.O. B o x 350, SF-O0101 Helsinki, Finland; tNeuropsychiatry Branch, National Institute o f Mental Health, N I M H Neuroscience Center at St. Elizabeths, Washington, D C 20032 R e c e i v e d 15 J u n e 1993; A c c e p t e d 4 F e b r u a r y 1994 HONKANEN, A., S. J. CHRAPUSTA, F. KAROUM AND E. R. KORPI. Alterations in dopaminemetabolism by intraperitoneal ethanol in rats selectedfor high and low ethanol preference: A 3-methoxytyramine study. ALCOHOL 11(4) 323-328 1994.- Effects of an ethanol dose (l g/kg, IP) on the metabolism of dopamine (DA) in the nucleus accumbens, striatum and hypothalamus of ethanol-naive alcohol-preferring (AA) and alcohol-avoiding (ANA) rats were studied. Rats were sacrificed by focused-beam microwave irradiation of the brain 20 minutes after ethanol administration, and the concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), assumed to reflect DA metabolism, and of 3-methoxytyramine (3-MT), assumed to reflect DA release, were measured using gas chromatography-mass spectrometry. Basal striatal DOPAC and HVA concentrations were higher in the AA rats in comparison with ANA rats. Ethanol increased HVA, but not DOPAC, concentration in the nucleus accumbens and striatum, hut not in the hypothalamus. There was a significant rat line x ethanol treatment interaction with respect to HVA concentration in the nucleus accumbens. The increase in HVA was higher in the AA than ANA rats. Basal 3-MT concentration was not changed by ethanol, except in the nucleus accumbens, where a significant rat line x ethanol treatment interaction was found. A decrease in 3-MT concentration was only detected in the ANA rats. After inhibition of monoamine oxidase with pargyline hydrochloride (75 mg/kg, IP, 10 min before sacrifice), 3-MT accumulation was decreased by ethanol, especially in the nucleus accumbens of both AA and ANA rat lines as well as in that of nonselected Wistar rats. The results suggest that 1) DA metabolism to DOPAC and HVA is dissociable from DA release as reflected by 3-MT production, 2) ethanol, if anything, reduces DA release, and that 3) the AA and ANA rats differ in their basal DA metabolism and in the ethanol effects thereupon, but not in ethanol-induced changes in DA release. Ethanol Dopamine metabolism Alcohol-avoiding rats

Nucleus accumbens

3-Methoxytyramine

P S Y C H O A C T I V E drugs abused by man share an ability to produce l o c o m o t o r stimulation in rodents, suggesting a link between this effect and the reinforcing properties of these drugs (54). The drug-related increases in locomotor activity apparently correlate with the activation of the mesolimbic dopamine (DA) projections, especially the pathway into the nucleus accumbens (NA) (7). Hence, dopaminergic activation produced by drugs of abuse may offer a useful measure of their addictive potential. In line with the above hypothesis, ethanol, a most com-

Alcohol-preferring rats

monly abused drug, has been reported to increase the firing rate of DAergic neurons in the ventral tegmental area and substantia nigra in vivo (15,50) and in vitro (3,50), suggesting either a direct action of ethanol on DAergic cells or disinhibition of DAergic cell function by a GABAergic mechanism (36). However, also local application of ethanol increases the release of D A in the nucleus accumbens and striatum (56,57). Therefore, there may be multiple mechanisms by means of which ethanol affects the DAergic systems. On the other hand, the neurochemical effects of ethanol on DAergic system have

To whom requests for reprints should be addressed. 323

324

HONKANEN ET AL.

often been contradictory. Ethanol has been shown to decrease (2), increase (12-14,21,26,28,39), or have no effects (20) on the turnover and metabolism of DA. Recent microdialysis investigations have suggested that ethanol may have a biphasic effect on DA release increasing it in the dose range of 0.252.5 g/kg IP while decreasing it at higher doses (4-5 g/kg) (22,57). Most of the above studies have utilized nonselected laboratory rodents. The comparison of rat lines selectively bred for differential alcohol preference (6) can also yield important information on the role of the DAergic system in the alcohol preference. Ethanol, given intragastrically, has been shown to increase DA metabolism more in various brain regions of Sardinian ethanol-preferring rats (sP) than in those of the nonpreferring (sNP) rats (13,14). Voluntary ethanol drinking increased extracellular DA more in nucleus accumbens in ethanol-preferring (P) rats than in nonselected Wistar rats (51). In contrast, no clear differences in elevation of the extracellular DA concentration by ethanol given intraperitoneaUy were observed between high-alcohol-drinking (HAD) rats and lowalcohol-drinking (LAD) rats (58). Alcohol-preferring A A (Alko, Alcohol) and alcohol-avoiding A N A (Alko, NonAlcohol) rat lines (10) have only minor differences in their basal levels of DA, its precursors and metabolites in the brain (1,30). DA metabolism appears to be sinu'larly stimulated by ethanol (2 and 4 g/kg, IP) in these rat lines (29). However, no information is currently available on the effects of ethanol on brain regional DA release in the A A and A N A rats. Here we report the effects of an intraperitoneal injection of ethanol on DA metabolism in the nucleus accumbens, striaturn and hypothalamus in the A A and A N A rats. DA metabolism was assessed by measuring 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. Concentration of 3-methoxytyramine (3-MT), D A metabolite that can only be formed from released DA, was used as a relative index of DA release (for review on the utility o f 3-MT measurements, see 55). METHOD

Animals Male adult APt and A N A rats from the generations F61 and F62, and H a n / W i s t a r rats (Department of Laboratory Animals, University of Helsinki) were maintained in stainless steel wire mesh cages in groups o f four to five animals under a 12/12 h light/dark cycle (lights on at 0600), at temperature of 22 ± 2°C and relative humidity o f 50 + 5070. The rats had free access to R3 pellet food (Ewos AB, S6dert~lje, Sweden) and tap water, and were accustomed to daily handling 4 to 5 times per week. Treatment Schedules and Brain Dissection In the first experiment, the A A and A N A rats were injected IP with either ethanol solution (12070 w/v in saline) at a dose o f 1 g/kg, or with an equivalent volume o f saline (control rats). The ethanol dose employed (1 g/kg) has been shown to produce robust stimulation of D A release in the nucleus accumbens of nonselected rats by microdiaiysis (22,57). It was also close to the amount that the alcohol-preferring A A animals voluntarily consume in a single drinking bout (44). Furthermore, this dose o f ethanol, but not lower ones, has produced conditioned place preference in nonselected rats (5), indicating it has reinforcing actions. Brain tissue samples from these rats were assayed for DOPAC, HVA, and 3-MT. Trunk

blood samples were collected into prechilled test tubes for the determination of ethanol levels. In the second experiment, the AA, ANA, and Wistar rats were first injected IP with ethanol (1 g/kg) or equivalent volume of saline as above. Ten min after the first injection, all rats received another IP injection containing the MAO inhibitor pargyline hydrochloride (Research Biochemicals Inc., Natick, MA) at a dose of 75 mg/kg to inhibit monoamine oxidase. This dose of pargyline has been shown to induce quick inactivation of MAO in the brain without yet affecting the DA synthesis during the first 10 min (18). Only 3-MT was determined from these samples. Twenty rain after the ethanol injection, each rat was taken from its cage to another room, killed immediately by microwave irradiation focused to the head (to prevent postmortem changes) using a model NJE 2603-10kW microwave instrument (New Japan Radio Inc., Japan) set at 6.5 kW output power for 1.3 s, and decapitated. The 20-min time point was chosen on the basis of previous findings, indicating that ethanors reinforcing and locomotor stimulating effects are evident only during the first 30 rain after administration (5,44,45). Brain was then removed from the skull, placed in a stainless steel brain mold, and sectioned with steel wires coronally at 2.7, -0.3 and -4.6 mm from the bregma (42). Nucleus accumbens (about 10 mg wet weight) and striatum (about 20 mg) were dissected from the second slice by using needles with inner diameters of 2 and 3 mm, respectively. Hypothalamus (about 40 mg) was dissected from the third slice by cutting along its upper and lateral borders. Determination o f DA Metabolites After the dissection, the brain regions were placed into 1.5-ml Eppendorf tubes on dry ice. Tissues were homogenized in 500 ~1 of 1M HCI containing deuterated 3-MT, DOPAC and HVA that were used as internal standards. Homogenates were centrifuged and supernatants stored at -800C until sent in dry ice from Helsinki to Washington, DC. Protein contents of the samples were assayed by a modification of the Lowry method (43). Analysis of the DA metabolite contents of the samples was carried out by mass spectrometry as described previously (9,24,25). Determination o f Blood Ethanol Trunk blood samples were centrifuged, and serum was separated and stored at -20°C until assayed for ethanol content by head-space gas chromatography (11). Statistics Two-way analysis of variance (ANOVA) was used to find significant rat line effects, ethanol treatment effects, and rat line x ethanol treatment interactions in metabolite concentrations, with Student's t-test as a post hoc test. RESULTS

DA Metabolite Concentrations After Ethanol Intraperitoneal injection of ethanol (1 g/kg) produced similar blood ethanol levels in the A A and A N A rats (24 ± 3 mM vs. 25 ± 3 raM, mean ± SEM, n = 8, respectively), but its effects on DA metabotite concentrations were different in the two rat lines (Table 1). In the nucleus accumhens, there were

ETHANOL EFFECTS ON DOPAMINE METABOLISM

325

TABLE 1 CONCENTRATIONS OF 3,4-DIHYDROXYPHENYLACETICACID (DOPAC), HOMOVANILLIC ACID (HVA) AND 3-METHOXYTYRAMINE(3-MT) IN VARIOUS BRAIN REGIONS OF ALCOHOL-PREFERRING AA AND ALCOHOL-AVOIDING ANA RATS AFTER ETHANOL ADMINISTRATION Concentration after Brain region

Rat line

Metabolite

N. accumbens

AA ANA AA ANA

DOPAC

AA ANA

Striatum

Hypothalamus

HVA

Saline 133 171 48 59

± + ± ±

15 II 4 4

3-MT

1.00 ± 0.05 1.31 ± 0.11

AA ANA AA ANA AA ANA

DOPAC

153 ± 16 119 ± 12 67 ± 5

AA ANA AA ANA AA ANA

DOPAC

HVA

HVA 3-MT

189 161 77 66

± ± ± ±

1.27 ± 0.08 1.38 ± 0.07

10 10 2§ 4§ 0.12 0.08

10.6 10.6 3.3 3.2 0.26 0.22

12.2 12.3 4.1 3.8 0.24 0.25

± ± ± ± ± ±

1.1 0.5 0.4 0.2 0,02 0,02

0.7 1.2 0.4 0.5 0.02 0.01

Xt, ET~ Xt

l.ll ± 0.05 1.00 ± 0.08§

± ± ± ± ± ±

± ± ± ± ± ±

ANOVA*

19 20 6¶ 5

163 133 79 68 1.47 1.37

54 ± 4

3-MT

Ethanol

RLt RL~t, ET$

Means + SEM (n = 7-8) are expressed in pmol/mg protein. *Two-way analyses of variance (ANOVA) were carried out for each brain region: RL, rat line effect; ET, ethanol treatment effect; and X, the interaction between them. tP 0.05, :~p < 0.01. §p < 0.05, ¶p < 0.01, for the difference from the corresponding value after saline (Student's t-test).

significant rat line × ethanol treatment interactions (p < 0.05) in the H V A and 3-MT concentrations, which were either increased by ethanol in the A A but not in A N A rats (HVA) or decreased in the A N A hut not in A A rats (3-MT). Nucleus accumbens D O P A C concentrations also tended to be increased by ethanol in the A A rats (rat line x ethanol treatment interaction F(1,26)= 3.89, p = 0.059; p < 0.05 for the difference from corresponding saline-treated A A controls). In the striatum, ethanol administration increased H V A but not D O P A C or steady-state 3-MT levels in both rat lines. Both D O P A C and H V A concentrations in the striatum were higher in the saline-treated A A than A N A rats. In the hypothalamus, the D A metabolite concentrations were similar for both rat lines and not affected by ethanol in either rat line.

Ethanol Effects on 3-Methoxytyramine Concentrations Following Pargyline This experiment was conducted to verify the rather unexpected lack o f increase in steady-state 3-MT concentration by ethanol. As shown in Fig. 1, ethanol did not increase 3-MT accumulation following pargyline in any o f the brain areas studied in the A A and A N A rats. On the contrary, the 3-MT accumulation was significantly (p < 0.05) decreased in both rat lines, being clearest in the nucleus accumbens. Ethanol significantly ( p < 0.05) decreased the 3-MT accumulation also in the nucleus accumbens o f nonselected ethanol-naive Wistar rats that were included in the second experiment. The 3-MT levels after saline and ethanol administrations were 3.1 _+ 0.4 and 2.5 :t: 0.3 p m o l / m g protein (mean + SEM, n = 6 and 8), respectively. No significant

ethanol effects were found in the striatum and hypothalamus o f Wistar rats (data not shown).

DISCUSSION The main finding of the present study was the acute ethanol-induced decrease in tissue 3-MT concentration following M A O inhibition with pargyline. This effect was observed in the ethanol-naive selectively bred alcohol:preferring A A and alcohol-avoiding A N A rats, as well as in nonselected Wistar rats. While this observation suggests that acute ethanol administration to ethanol-naive rats induces a suppression of D A release, the changes in the H V A , and to lesser extent in D O P A C , concentration found in the A A and A N A rats indicate that D A metabolism was stimulated by ethanol in the nucleus accumbens and striatum. This latter finding may be attributed to stimulation of D A synthesis a n d / o r turnover. The finding that ethanol increased D A metabolism more in the alcohol-preferring A A rats than alcohol-avoiding A N A rats is consistent with results obtained with alcohol-preferring sP and nonpreferring sNP rats (14). 3-MT responses have not been studied in these rat lines. There may be several possible reasons for the dissociation between the concentration changes of 3-MT and the acidic metabolites (i.e., between D A release and metabolism, respectively). It should be noted, however, that it is the blunted synaptic release o f DA, not its enhanced intraneuronal metabolism, that correlates with previous behavioral findings, i.e., the absence of locomotor stimulation in the A A and A N A rats by the same IP dose o f ethanol (44). However, as studies conducted

326

HONKANEN E T AL.

n. accumbens

5 4 t

3

V//A I//A ////11

2


EL

E

0

o

E o_

striatum

10

r---

E

8 m

x

6

Ei

4

o r--

o

c

2

~c

0

c

2.0

o

o 0

1.5

hypothalamus

-1-

1.0

0.5

AA rats

ANA rats

FIG. 1. Effect of ethanol on concentration of 3-methoxytyramine in various brain regions of alcohol-preferring (AA) and alcohol-avoiding (ANA) rats following monoamineoxidas¢ inhibition.Ethanol (1 g/kg, IP; striped bars) or saline (5 ml/kg; open bars) was administered 20 min and pargyline (75 mg/kg, IP) 10 rain before sacrifice by focused microwave brain irradiation. Results are means + SEM (n = 5-6). ANOVA: ethanol effect for nucleus accumbens F(I, 18) = 14.6, p = 0.0012; for striatum F(I, 18) = 7.58, p = 0.013; for hypothalamus F(I, 18) = 5 . 6 1 , p -- 0 . 0 2 9 . No significant rat lineeffects or rat line x ethanol treatment interactions. *p < 0.05 for the significance of the difference from corresponding saline-treated group (Student's t-test).

with ~-opioid agonists have shown, dissociation between changes in locomotor activity and DA transmission can occur (8), which makes the above correlation only suggestive. A number of studies have shown activation of DA metabolism by various doses of ethanol (for references, see Introduction), but only three studies exist on 3-MT determinations (without microwave irradiation). Liljequist and Carlsson (32) found that ethanol (2.36 g/kg, IP) in pargyline-treated mice decreased the 3-MT formation in whole brain. Hunt and Majchrowicz (21), giving nonpargyline-treated rats a high oral dose (6 g/kg) of ethanol, observed that striatal DOPAC and HVA concentrations increased while 3-MT concentration did not. Recently, Milio and Hadfield (37) reported that 3.5 g/kg ethanol (IP) decreased 3-MT concentration in striatum and olfactory tubercle in nonpargyline-treated mice, while striatal DOPAC and HVA levels were increased. These results are in agreement with the idea that in the presence of ethanol the DA metabolism and release may be dissociated, even if higher ethanol doses were used in these studies than in the present study. There is a sharp contradiction between the 3-MT results of the present study and the microdialysis results, in which ethanol has consistenly increased extracellular DA levels (22,58). At present, there may be several reasons for this discrepancy. First, stressful experiencies have been shown to sensitize mesolimbic DA neurons to the stimulating actions of subsequent drug administration (23). Therefore, it might be possible, that the obvious stress during the invasive procedure of intracerebral dialysis probe implantation including anaesthesia, sensitizes to some degree the DA mechanisms to stimulatory actions of ethanol even without any prior ethanol experience. Second, the utility of the steady-state tissue 3-MT measurements as indices of DA release has been questioned, particularly when an increase in DA release may occur, e.g. after neuroleptics (4,53). More recently, increases in steady-state tissue 3-MT levels by haloperidol have been found when an improved microwaving instrument was used (24). Tissue 3-MT concentration, particularly following pargyline administration, is indeed widely accepted as a good indirect index of relative DA release, both when decreases or increases in the release occur (55). Moreover, 3-MT formation has also been postulated to reflect receptor-relevant synaptic DA levels more accurately than dialysable DA concentrations (31), supported by a recent study indicating that the postsynaptic membranebound catechol-O-methyltransferase (COMT), and not its glial counterpart, is primarily responsible for the formation of tissue 3-MT (40,41). Based on these facts, the ethanol-related decrease in the nucleus accumbens' 3-MT concentration found in the pargyline-treated ANA rats is consistent with the decrease in the nucleus accumbens' steady-state 3-MT level in these rats. The absence of significant ethanol-related changes in the steady-state 3-MT levels of the AA rats and other brain regions of the ANA rats is also compatible with the acute ethanol-related reductions in the respective 3-MT levels seen after pargyline, since tissue 3-MT level after pargyline is, in general, a more sensitive and accurate index o f changes in DA release than steady-state tissue 3-MT level (55). Third, ethanol affects the function of some membranebound enzymes (48). MAO activity is decreased by ethanol (49), indicating that the decreased 3-MT accumulation by ethanol is not due to abolishment of pargyline-induced MAO inhibition by ethanol. 3-MT is not conjugated in the brain (52) and it does not use the probenecid-sensitive transport system as the acidic metabolites do (19), which makes its clearence from the brain very slow. Therefore, it is possible that the

ETHANOL EFFECTS ON DOPAMINE METABOLISM decreased tissue 3-MT content resulted from inhibitory action of ethanol on DA uptake mechanism, needed to transfer DA to COMT site, or on COMT activity. If so, inhibition of these systems would at least partly explain also the increase of extracellular DA after local application of ethanol into striatum (56,57). However, at present there are no published data on the influence of ethanol on COMT, while one study reports that subchronic ethanol treament (24-h inhalation of ethanol vapor) has no effects on [3H]DA accumulation in striatal synaptosomes (38). Fourth, COMT activity has been shown to be inhibited in vivo by condensation products of DA and either ethanol metabolite acetaldehyde (salsolinol) or 3,4-dihydroxyphenylacetaldehyde (tetrahydropapaveroline) (17). However, these authors could demonstrate the inhibition of COMT activity by acute ethanol only with a high oral dose (5 g/kg) combined with L-dopa administration. Therefore, the possibility that condensation products are also involved in the decrease of tissue 3-MT levels by the low dose of ethanol (1 g/kg) observed in the present study seems rather unlikely. At the neurobiological level, there are many other possibilities by which ethanol could modulate DAergic system. Ethanol has been shown to interact with 7-aminobutyric acidA, N-methyl-D-aspartate, and serotonin 5-HT 3 receptors (33,34, 47). Ethanol also interacts with neurotensin, cholecystokinin, and endogenous opioid (16,27,35). All these interactions can alter the activity of DAergic system indirectly. For example, like ethanol, benzodiazepine agonists enhance GABAA mechanisms, and benzodiazepine application is associated with diminished DA release in the nucleus accumbens (59). It remains to be studied whether any of these indirect actions of ethanol are involved in the dissociation of 3-MT and acidic metabolite changes.

327 The nucleus accumbens has been implicated in the rewarding mechanisms of various drugs, being the main site where drug-related changes occur in the DA release (7). It is interesting that the same brain region may also be involved in the aversive mechanisms (46). Therefore, it is plausible that the acute dose of ethanol to ethanol-naive animals activates only the aversive mechanisms, even in alcohol-preferring rat line, which might then have the biochemical correlate in decreased DA release. During chronic administration (5,45) or during voluntary drinking (44), tolerance can be produced to these aversive effects, thus revealing the real rewarding potential of ethanol. Therefore, acute studies on the rewarding potential of ethanol remain questionable. In summary, acute ethanol administration increased the levels of presynaptically formed DA metabolites in brain of both alcohol-preferring and alcohol-avoiding rats. In contrast, after MAO blockade, the concentration of 3-MT, DA metabolite formed outside the DA neurons, was decreased in both rat lines, suggesting decreased DA release. The decreased DA release by IP ethanol even in selectively bred alcoholpreferring rats suggests that ethanol administration may be aversive to ethanol-naive animals. Further work will be needed to reveal the mechanisms underlying the decreased 3-MT formation, and how it is presumably reversed as the alcoholpreference is developing in the AA rats, in which voluntarily ingested ethanol is reinforcing (44). ACKNOWLEDGEMENTS The authors wish to thank Pirkko Johansson and Randy Alexander for expert technical assistance, and Petri Hyyti~iand Pekka T. M~innist6for valuable comments on the manuscript.

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