Oral ethanol self-administration in rats is reduced by the administration of dopamine and glutamate receptor antagonists into the nucleus accumbens

Psychopharmacology (1992) 109:92-98 Psychopharmacology © Springer-Verlag 1992

Oral ethanol self-administration in rats is reduced by the administration of dopamine and glutamate receptor antagonists into the nucleus accumbens Stefanie Rassnick, Luigi Pulvirenti, and George F. Koob

Department of Neuropharrnacology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA

Received October 22, 1991 / Final version March 30, 1992

Abstract. The purpose of this study was to assess the role of endogenous dopamine and glutamate systems within the nucleus accumbens in modulating responses for oral ethanol reinforcements (10% w/v) in a free-choice operant task. Pretreatment with both systemic (100 lag/kg) and intra-nucleus accumbens microinjection of fluphenazine (2 and 4 lag), a dopamine receptor antagonist, significantly decreased responding for ethanol, without significantly affecting responses for water. Ethanol self-administration was also attenuated by microinjection into the nucleus accumbens of 2-amino-5-phosphopentanoic acid (AP-5, 3 and 6 lag), a competitive NMDA receptor antagonist. These results suggest that dopamine and glutamate neurotransmission in the nucleus accumbens may regulate ethanol self-administration and its reinforcing effects.

Key words: Ethanol self-administration - Dopamine antagonist - NMDA antagonist - Nucleus accumbens

Research towards understanding the basis of ethanol (EtOH) abuse and alcoholism supports a multidimen- sional model that includes biological, genetic and psycho- social variables (i.e., neurochemical mechanisms, family history, prior experience and environment) (Cloninger 1987) to explain why EtOH abuse continues to be a major health and social problem in society. These factors may be interrelated but not necessarily interdependent variables, contributing to the regulation processes associated with the tendency to abuse EtOH.

EtOH-seeking behavior and abuse may occur since EtOH reduces tension or anxiety (Cappelt and Herman 1972) and produces euphoria (Kornetsky et al. 1988), and these properties may be linked to EtOH self-administration. Biological studies have focused on neuronal mechanisms as an essential component in the regulation of EtOH intake and its reinforcing effects. However, the precise neurochemical mechanisms underlying the reinforcing properties of EtOH are still unclear. Research suggests that the mechanisms mediating EtOH self-administration

Correspondence to: G.F. Koob

involve at least dopamine (Pfeffer and Samson 1988), norepinephrine (Brown and Amit 1977; Corcoran et al. 1983), GABA (Boismare et al. 1984; Samson et al. 1987; Hwang et al. 1990), opioid peptides (Reid and Hunter 1984; Mudar et al. 1986; Hubbell et al. !991) and serotonin (Geller 1973; Rockman et al. 1982; Murphy et al. 1987) neurotransmission.

Neurochemical, electrophysiological and behavioral evidence suggests that dopamine transmission may play an important regulatory role in reinforcing properties associated with EtOH intake. EtOH is known to stimu- late dopamine activity within mesolimbic-forebrain areas. In vitro and in vivo studies show that EtOH stimulates firing of A10 dopamine neurons in the ventral tegmental area (VTA) (Gessa et al. 1985; Brodie et al. 1990) and also increases extracellular dopamine release within the nucleus accumbens (N.Acc.) (Imperato and Dichiara 1986; Wozniak et aL 1991; Yoshimoto et al. 1991), a projection area of the dopaminergic cell bodies of VTA. Voluntary EtOH intake may also activate the mesolimbic dopamine system (Fadda et al. 1989).

While previous research has suggested that the activ- ating and reinforcing effects of other drugs of abuse (such as psychostimulants and opiates) may involve the N.Acc. of the ventral striatum as a critical neural substrate (Koob and Bloom 1988), the role of neurochemical substrates in the nucleus accumbens for EtOH reinforcement has not been extensively studied. To examine the functional importance of dopamine in modulating EtOH-seeking behavior, fluphenazine, a dopamine receptor antagonist, was administered systematically and also microinjected into the N.Acc. in rats trained to self-administer EtOH. An animal model of voluntary oral EtOH self-administration was used where rats were trained to self-administer EtOH (10% w/v) or water by responding at one of two levers in a free-choice operant task. This procedure was modified from Samson (1986), and allows for an assess- ment of the acute reinforcing effects of limited access to EtOH without producing physical dependence on EtOH.

Along with dopaminergic projections to the N.Acc, this nucleus receives major afferent connections from allocortical areas (Kelly and Domesick 1982; Kelly et al.

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1982). Anatomica l studies indicate that these afferents are glutamatergic in na ture (Fuller et al. 1987), and may originate in the amygdalo id complex (Christie et al. 1987) and h ippocampal format ion (Walass and F o n n u m 1979). N.Acc. glutamatergic afferents may partially overlap with other dopaminergic limbic afferents innerva t ing the N.Acc., such as those arising from the VTA (Kelly et al. 1982). Fur thermore , biochemical and behavioral studies (Hami l ton et al. 1986) suggest a facilitatory role for glutamate on dopamine activity within the N.Acc.

These results p rompted an examina t ion of the func- t ional role of glutamate afferents to the N.Acc. in mediat ing E t O H self-administration. To examine the importance of g lutamate receptors within the N.Acc. in mediat ing E t O H reinforcement, the effects of microinject ion of 2 -amino-5-phosphopentanoic acid (AP-5), a competit ive N M D A receptor antagonist , into the N.Acc. was tested in rats t ra ined to orally self-administer E tOH. The N.Acc. is known to conta in N-methyl-D-aspartate (NMDA) receptors ( M a n a g h a n et al. 1988), one receptor subtype that mediates glutamate neuro t ransmiss ion (Watkins and Mogenson 1981).

13, 5% EtOH without saccharin was available. Thereafter, the concentration of EtOH was increased to 8%, with 2 days of access to an EtOH-saccharin solution and 1 day of access to 8% EtOH without saccharin. EtOH at 10% then was introduced in the pres- ence of saccharin, and training to respond for this concentration was conducted for 2 days.

Animals were then trained to respond for 10% EtOH and water in the free-choice task. On baseline and testing days, responding for 10% EtOH and water was conducted in the absence of water and food deprivation, and without sweeteners in the EtOH drinking solution. Responding for EtOH was defined as stable when responses were +_ 20% for 3 consecutive days. All training and testing sessions consisted of 30-min daily sessions conducted between 1200 and 1500 hours.

Sur#ical procedure for intracerebral cannulation. Following the es- tablishment of baseline responding, rats were anaesthetized with halothane and stereotaxically (Kopf instrument) implanted with bilateral 23-gauge, 10-mm steel intracranial cannulae aimed 3 mm above the N.Acc.. Stereotaxic coordinates were based on the atlas of Pellegrino and Cushman (1979): anterior + 3.2 mm and lateral + 1.7 to bregma. The dorsal/ventral coordinate was -4 .8 mm

from the skull surface. Cannulae were fastened to the skull with dental cement and sealed with a 10-mm stylet wire. Animals were allowed a minimum of 3 days recovery before being reintroduced into the EtOH free-choice operant task.

Materials and methods

Subjects. Male Wistar rats (Charles River laboratory) weighing 220-240 g at the beginning of the experiments served as subjects. Rats were water deprived for 4 days only (22 h/day) to motivate responding in the operant chambers during the first four training sessions. Thirty minutes after the training sessions for those 4 days only, water was available for 1 h in the home cage. Food and water were then available ad libitum throughout subsequent training and testing periods.

Behavioral testino apparatus. Behavioral testing was conducted in operant chambers (Coulbourn Instruments) located in sound-attenuated cubicles equipped with exhaust fans. The fluid delivery system in each operant chamber consisted of two containers (for EtOH solutions and water) connected to solenoids and two adjacent stainless steel drinking cups (volume capacity: 0.15 ml) mounted 4 cm above the grid floor and centered on the front panel. Two retractable levers were located to either side (4.5 cm) of the drinking cups. Responses at one of the two levers delivered EtOH solution (0.1 ml/response) or the same quantity of water to the corresponding cup. A ceiling light in each chamber turned off to indicate the onset of the session. Recording of responses and fluid delivery was control- led by a microprocessor (SPIDER) using SPIDER software (Paul Fray Ltd., Cambridge, England).

Procedure. Animals were trained to orally self-administer EtOH using a variant of the sucrose fading technique previously described by Samson (1986). In the present study, saccharin was added to the EtOH solution (instead of sucrose) to increase the palatability of the EtOH solution and to overcome EtOH's aversive taste. Initially, rats were trained for 3 days in 30-min daily sessions to respond on either of two operant levers for 0.2% (w/v) saccharin reinforcements on a fixed ratio-1 (FR-1) schedule. Then rats were trained on a free- choice task where responses at one lever delivered response-contin- gent EtOH-saccharin (0.1 ml/response) and responses at the other lever resulted in the delivery of the same quantity of water, with the EtOH side alternated daily. In the free-choice task all responses were reinforced on a FR-1 schedule of reinforcement. During training days 4-9, rats were trained to respond for 5% (w/v) EtOH solution containing saccharin (0.2% w/v) or water. On training day 10, rats were given access to 5% EtOH and water. On days 11 and 12, the 5% EtOH-saccharin solution was reintroduced, and on training day

Drug administration. EtOH for oral self-administration was prepared from 100% ethyl alcohol and diluted with tap water for concentrations of 5, 8 and 10% (w/v). Fluphenazine dihydrochloride and 2 amino-5-phosphopentanoic acid (AP-5) (Research Bio- chemicals, Inc.) were dissolved in 0.9% sodium chloride (saline) and prepared immediately before administration. Fluphenazine (0, 10, 30 and 100 Bg/kg) was administered by intraperitoneal (IP injection with a 2-h pretreatment interval before the test session. For intracerebral injections, fluphenazine and AP-5 were administered using a microinjection technique with pieces of calibrated polyethy- lene tubing that were connected to 10-lal Hamilton syringes and a Harvard infusion pump. For microinjections into the N.Acc., rats received bilateral infusions (1.0 pl over 2.05 min) of saline vehicle and fluphenazine (2.0 and 4.0 lag) or AP-5 (3.0 and 6.0 lag) through 30-gauge injectors inserted to extend 3 mm beyond the ventral tip of the cannulae. Prior to the first drug injection, rats were habituated to the injection procedure with the insertion of dummy 10-mm injectors. Saline vehicle was administered as the first injection; thereafter, the order of drug injections was counterbalanced using a Latin square design between all animals.

Experimental design and data analysis. Responding for EtOH was defined as stable when responses were + 20% for 3 consecutive days. For each animal, baseline was computed as the average response for the 4 days prior to saline vehicle administration. In each experiment, a within-subjects Latin square design for drug administration was used. Each experiment was conducted with a separate group of rats serving as subjects: systemic fluphenazine experiment, n = 12 rats; intra-N.Acc, fluphenazine, n = 7 rats; and in the intra- N.Acc. AP-5 experiment, n = 8 rats. Responses for EtOH and water were expressed as percent of baseline responding and analyzed using separate one-way analyses of variance (ANOVA) each with a within- subjects factor for dose. Differences among individual means were subsequently determined by Newman-Keurs post-hoc test. The average total number of responses (EtOH plus water) and the average EtOH preference score (percent of responses for EtOH relative to the total number of responses) were also computed for each experiment. To control for possible skewed distribution due to the transformation of the data to preference (proportion) scores, all ANOVAs of the proportion scores were recalculated using the arcsin transformation (Winer 1971). Only those analyses showing a difference between the non-arcsin transformed and the arcsin transformed analyses are reported.

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Blood EtOH concentrations. To verify blood EtOH content, blood samples were obtained immediately after a 30-min session of access to 10% EtOH in the free.choice task on a baseline training day. A sample of mixed arterial and venous blood was obtained by tail-bleed method. Samples were assayed for blood EtOH content using the NAD-ADH enzyme spectrophotometric method (Sigma Biochemicals).

Histology. At the completion of the experiment, animals were sac- rificed with an overdose of pentobarbital and perfused intracar- dially, first with saline and then with a formalin/saline solution. The brains were subsequently removed from the skull, frozen and sec- tioned at 50 microns. Mounted sections were stained with cresyt violet. Injection sites were verified under a light microscope.

Results

As shown in Fig. 1, systemic fluphenazine pretreatment significantly reduced responding for EtOH (ANOVA

150 -

Responding for EtOH 10% (w/v)

c

CO

100

50

-T -

10 30

Fluphenazine (~g/kg)

-1-

100

tso] Responding for Water

a) lOO

~ 50

o o lO 30 lOO

Fluphenazine (l.tg/kg)

Fig. i. Effects of systemic fluphenazine on responding for EtOH and water in the free-choice task. Animals had limited access to EtOH (10% w/v) and water in the free-choice EtOH self-administration 30-min operant task. Data have been expressed as percent of baseline responding (mean + SEM) and are plotted as a function of dose fluphenazine. Asterisk indicates a significant difference for responding as compared with responding after vehicle injection. **P < 0.01, Newman-Keul's a posteriori test

main effect: (F(3,33) = 8.27, P < 0.001]), without producing a significant effect on responding for water [F(3,33) = 2.38, NS]. At the time of testing, the average ( + SEM) baseline response for EtOH was 31.73 +_ 1.45 and average ( _+ SEM) baseline responses for water were 20.28 +_ 2.83. When EtOH preference scores were analyzed, EtOH preference was significantly different after 100 ~g/kg fluphenazine [F(4,44) = 4.4, P < 0.005, New- man-Keuls test P < 0.05 as compared with baseline and saline]. Total responses (ethanol plus water) averaged ( + SEM): 52 _+ 3.6 for baseline testing conditions; 54.6 +_ 4.7 after saline; 46 _+ 5.1 after 10 t, tg/kg; 36.7 ___ 7.3 after 30 [ag/kg; and 25.8 _+ 4.4 after 100 ~tg/kg systemic fluphenazine. Average ( +__ SEM) E t O H preference scores were 62.7 + 2.9% for baseline testing conditions; 65.1 +__4.5% after saline injection; 55.2 + 4 . 0 % after 10~tg/kg; 66 + 3.2% after 30~tg/kg; and 45.2 + 5.3% after 100 lag/kg systemic fluphenzaine.

Microinject ion of f luphenazine into the N.Acc. immediately before the test session selectively reduced responding for E t O H (Fig. 2) [F(2,12) = 12.18, P < 0.005]

Responding for EtOH 10% (w/v)

150 -

100

50

~,,, , I 0 pg 2 Bg

Fluphenazine

--T--

4 Bg

150

Responding for Water

1oo e-

N 50

0 0~t0 2~g 4~0

Fluphenazine

Fig. 2. Effects of microinjection of fluphenazine into the N. Acc. on responding for EtOH and water. Data have been expressed as percent of baseline responding (mean + SEM) and are plotted as a function of dose of fluphenazine. Asterisks indicate significant difference for responding as compared with responding after vehicle injection.**P < 0.01, Newman-Keul's a posteriori test

without altering responding for water [F(2,12)= 1.66, NS]. For this group of animals, average ( + SEM) baseline responses for EtOH and water were 33.14 + 4.5 and 15.38 + 3.36, respectively. When EtOH preference scores were analyzed, EtOH preference was significantly different following 2 lag intra-N.Acc, fluphenazine [F(3,18)= 6.0, P < 0.05, Newman-Keuls test P < 0.01, compared to baseline and saline]. Total responses (ethanol plus water) averaged ( + SEM): 48.5 +__ 5.5 for baseline conditions; 46 ___ 7.4 after saline; 27.1 _ 3.7 after 2 lag; and 25.1 ___ 4.3 after 4 lag. Average ( + SEM) EtOH preference scores were 67.8 + 2.6% for baseline, 63.0 +__ 5.9% after saline; 36.1 + 6.5% after 2 lag and 56.6 ___ 8.2% after 4 lag intra- N.Acc. fluphenazine injection.

EtOH self-administration was also reduced by microinjection of AP-5 into the N.Acc., as illustrated in Fig. 3. ANOVA showed that AP-5 selectively attenuated responding for EtOH (main effect: [F(2,14)= 5.23, P < 0.05, Newman-Keul's A posteriori test] without altering responding for water [F(2,14) = 0.037, NS]. Average base-

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line EtOH and water responses were 28.77 + 4.2 and 22.29 ___ 2.6, respectively. When ethanol preference scores were analyzed, E tOH preference was not significantly different from baseline responding after any dose of AP-5 [F(3, 21) < 1]. Total responses (ethanol plus water) averaged ( + SEM): 51.1 ___ 5.5 for baseline, 47.1 + 6.7 after saline; 39.1 ___ 7.6 after 3 lag; and 36.2 + 4.9 after 6 lag. Average ( + SEM) EtOH preference scores were 55.5 + 4 . 4 % for baseline; 6 3 . 0 + 6 . 6 % after saline: 59.1 ___ 9.9% after 3 lag; and 52.9 + 9.5% after 6 lag intra N. Ace. AP-5 injection.

The site of injection into the N. Ace. for each animal is shown in Fig. 4. Cannulae placements for injection were verified within the region of the N. Acc. from 3.0 to 3.8 mm anterior to bregma.

Average ( + SEM) blood EtOH concentrations determined immediately after a baseline session were 37.8 __+ 7.3 mg% for the subjects participating in the sys- tematic fluphenazine experiment; 44.2 + 14.5 mg% and 28.8___ 4 .4mg% for subjects in the intra-N.Acc. fluphenazine and AP-5 experiments, respectively.

150

Responding for EtOH 10=/= (w/v )

L-

== ca 1=3

100

50,

- r -

0 p,g 3 p.g 6 tag

AP-5

150

Responding for Water

o 100 e,,

L13 nm

N 5O

o o Bg 3 ~g 6 ~g

AP-5

Fig. 3. Effects of microinjection of AP-5 into the N.Acc. on responding for EtOH and water in the free-choice task. Data have been expressed as a percent of baseline responding (mean + SEM) and are plotted as a function of dose AP-5. Asterisks indicate significant difference for responding as compared with responding after vehicle injection. *P < 0.05, Newman-Keui's a posteriori test

Discussion

The results reported here suggest that E tOH self-administration is related in part to dopamine and glutamate neurotransmission within the N.Acc. In the two-lever, free-choice operant task, EtOH-reinforced responding was significantly reduced by both systemic and intra- N.Acc. injection of fluphenazine, while responses for water were not significantly altered. EtOH self-administration was also attenuated by infusion into the N.Acc. of AP-5, a competitive NMDA receptor antagonist, indicating that glutamate and dopamine mechanisms within the N.Acc. may modulate responding for EtOH.

To further analyze the selectivity of the reductions in EtOH self-administration by the drug treatments, the raw data for each experimental session were transformed to percent of total fluid intake for that session and re- analyzed. This analysis revealed a significant decrease in preference for EtOH over water at t00 lag/kg systemic fluphenazine and 2 lag N.Acc. fluphenazine. None of the AP-5 doses showed a significant decrease in EtOH preference, suggesting a less selective action for AP-5. However, it should be noted that the effect of drug treatments on responding for water in this paradigm is much more variable than responding for EtOH and appears to be largely associated with EtOH responding. There is no

3.0 3.2 3.4 3.6 3.8

Fig. 4. Histological localization of cannulae placement for all animals in the intracerebral experiments

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explicit motivation to respond for water other than a thirst associated with drinking EtOH, since the animals were not water or food deprived. Thus, weak drug effects on EtOH responding can be masked by high variability in the preference scores.

The attenuation of responding for EtOH by systemic flupI~enazine treatment is consistent with previous studies demonstrating that voluntary EtOH intake in rats is reduced by antagonists of dopamine receptor activity, such as haloperidol, a post-synaptic D-2 receptor antagonist (Pfeffer and Samson 1988), and gamma-butyrolactone, a drug that reduces dopaminergic cell firing (Fadda et al. 1983).

Intra-N.Acc. infusion of fluphenazine also attenuated responding for EtOH, suggesting that dopaminergic activity within the N.Acc. modulates the reinforcing effects of EtOH. The inhibition of EtOH self-administration by intra-N.Acc, fluphenazine was not dose dependent, which may reflect a more contributory rather than an essential role for dopamine in EtOH reward.

Responding for EtOH and total EtOH is also decreased when the baseline dose of EtOH available per reinforcement (10%) is decreased by 50% (Samson 1986; Rassnick and Koob, unpublished observations). This suggests that a reduction in the relative efficacy of EtOH reinforcement is manifested as a decrease in the total number of responses for EtOH. Thus, oral self-administration of EtOH differs from intravenous cocaine or heroin in that a compensatory increase in responding for intravenous cocaine or heroin occurs when the baseline dose available per reinforcement is reduced (Koob and Goeders 1989). Thus, the present results provide evidence for a role of dopamine systems as one of many mechanisms contributing to EtOH self-administration.

A role of dopamine for the reinforcing effects of EtOH is consistent with the previously described facilitatory effects of EtOH on dopaminergic neuronal activity. Bio- chemical studies indicate that EtOH increases dopamine activity in mesolimbic-forebrain areas. EtOH produces a dose-dependent increase in the spontaneous firing rate ofdopamine neurons in the ventral tegmental area (Gessa et al. 1985; Brodie et al. 1990). EtOH also increases extracellular dopamine release from the N.Acc. (Imperato and DiChiara 1986; Wozniak et al. t991; Yoshimoto et at. 1991), a dopamine terminal region of the VTA. Based on these studies indicating that acute EtOH treatment increases dopamine activity and a report that voluntary oral EtOH administration produces appreciable brain EtOH concentrations shortly after intake as measured by in vivo microdialysis (Ferraro et al. 1991), EtOH reinforcement may be mediated, in part, by an increase in dopamine neurotransmission.

Although there is some controversy concerning the specific involvement of dopamine systems in mediating EtOH reinforcement, much evidence exists to show that ethanol drinking is resistant to disruption of dopamine function (Brown et al. 1982; Linesman 1990). Systemic dopamine antagonists have been shown to have no effect on oral EtOH intake over a 24-h period (Brown et al. t982) or on EtOH drinking in water-deprived rats given 1 h limited access to a sweetened EtOH solution and water (Mudar et al. 1986), and to decrease both EtOH and

water drinking (Linesman 1990; Hubbell et al. 1991). It is interesting that manipulations of the dopamine system seem most effective in situations involving the use of operant (lever pressing) responding for oral EtOH and less effective when drinking (the consummatory response) is the dependent measure of EtOH reward (Brown et al. 1982; Mudar et al. 1986). That the relative importance of a given neurotransmitter system may depend on the na- ture of the response for the reward has precedence in theorizing about the biological basis of reward (Glickman and Schiff 1967).

Dopamine systems have been hypothesized to be an "intermediate critical link" in all rewards (Wise and Rompre 1989) including natural rewards such as food (Wise et al. 1978) and water (Gerber et al. 1981). Others have argued for dopamine-independent as well as dopamine-dependent contributions to drug reward such as for the opiate systems (Koob 199t). EtOH reward may involve multiple neurotransmitter systems with some con- tribution of the mesolimbic dopamine system in non- dependent animals working for low EtOH doses.

EtOH self-administration was also attenuated by microinjection of AP-5, a competitive NMDA receptor antagonist, into the N.Acc., suggesting that NMDA receptors in the N.Acc., possibly from limbic afferents, may modulate responses for EtOH. These results suggest that N.Acc. is a neural substrate that may be involved in the reinforcing effects of EtOH, and indicate that the integra- tive output of the N.Acc. may be modified by glutamate, dopamine and possibly other neurotransmitters that are either intrinsic or project to this region.

Considering that glutamate receptors are heterogen- ous and found in many brain regions (Cotman et al. 1987), the functional importance of activation of these receptors may display regional specificity. The N.Acc. glutamatergic afferents may originate in the amygdaloid complex (Christie et al. 1987) or hippocampal formation (Walass and Fonnum 1979). Autoradiographic studies show that the amygdala and hippocampus contain a moderate den- sity of NMDA receptor sites (Maragos et al. 1988) and also utilize glutamate as a transmitter for projections to the N.Acc. (Walaas 1981; Christie et al. 1987; Fuller et al. 1987). A glutamatergic pathway from the medial prefron- tal cortex to the N.Acc. has also been proposed (Carter 1980; Christie et al. 1985), but the specific receptor sub- types which mediate this projection have not been studied.

While the direct influence of allocortical glutamate neurons upon dopamine activity within the N.Acc. has not been investigated, the N.Acc. has been suggested to respond to (Callaway et al. 1991) and modulate the effects ofamygdala stimulation (Yim and Mogenson 1983, 1989). For example, inhibition of spontaneous locomotor activity which occurs following amygdala glutamatergic stimulation can be reversed by low doses of dopamine administered into the N.Acc. (Yim and Mogenson 1989).

The functional interdependence of glutamate and dopamine systems in the N.Acc. has also been studied. A facilitatory rote for glutamate on dopamine activity in the N.Acc. has been suggested based on biochemical studies showing that glutamate agonists release dopamine from accumbal slices, an effect which is inhibited by glutamate antagonists, and behavioral studies demonstrating

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that the hyperactivity elicited by injection of excitatory amino acids into the N.Acc. can be at tenuated by systemic reserpine or fluphenazine injection into the N.Acc. (Donzant i and Uretsky 1983). Other studies show that an tagonism of N M D A function within the N.Acc. can suppress the hyperactivity produced by intra-N.Acc. dopamine (Hamil ton et al. 1986; Pulvirenti et al. 1991), systemic cocaine (Pulvirenti et al. 1991), and amphetamine (Hutson et al. 1991), These results suggest that glutamate neurotransmission may modulate the output of the N.Acc.

The N. Acc. has been described as an integrator of sensory input and motor-re la ted output (Mogenson et al. 1982). Besides receiving dopaminergic and glutamatergic inputs, the N.Acc also contains GABA-ergic efferent projections to effector areas (i.e., ventral pallidum) (Swerdlow and K o o b 1984). The N.Acc. comprises part of a neuronal circuitry impor tant for drug reward, previously described as an allocortical-limbic-accumbens-pallidal system (Koob and Bloom 1988). Al though E t O H may act at different points in this system through multiple neurotransmitter systems, the present results suggest a role for dopamine and glutamate at the level of the N.Acc.

In summary, the results of this study show that administration of dopamine and N M D A receptor antagonists into the N.Acc. decreased responses for E t O H during limited access to E t O H and suggest that dopamine and glutamate may play a role in the neurochemical mediat ion of the acute reinforcing effects of EtOH. E t O H intake may be modula ted by dopamine mechanisms within the N.Acc,, and it is possible that N M D A receptors may regulate E t O H self-administration by influencing dopaminergic mechanisms within the N.Acc. These find- ings also extend the role of the N.Acc. as an anatomical substrate comprising part of the neural circuitry that modulates the mildly intoxicating/reinforcing properties of E t O H as well as psychomotor stimulants and opiates.

Acknowledgements. This research was supported in part by the National Institute on Alcohol Abuse and Alcoholism grants: AA 08459, awarded to G.F. Koob; a predoctoral research fellowship award, AA 05297, to S. Rassnick; and Alcohol Center Grant AA 06420, Floyd E. Bloom, Director. This work was also supported in part by grant provided by the Alcohol Beverage Medical Research Foundation to G.F. Koob and a research fellowship award from SANDOZ, Basel to L. Pulvirenti. The authors gratefully acknow- ledge Mr. Robert Lintz for his excellent technical assistance. We thank Molecular and Experimental Medicine's Word Processing Center for assistance in manuscript preparation. This is publication number 7064-NP of the Scripps Research Institute.

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