Regulate Alcohol Drinking Research Articles

GABA release from central amygdala neurotensin neurons differentially modulates ethanol consumption in male and female mice.

The central nucleus of the amygdala is known to play key roles in alcohol use and affect. Neurotensin neurons in the central nucleus of the amygdala have been shown to regulate alcohol drinking in male mice. However, little is known about which neurotransmitters released by these cells drive alcohol consumption or whether these cells drive alcohol consumption in female mice. Here we show that knockdown of GABA release from central amygdala neurotensin neurons using a Nts-cre-dependent vGAT-shRNA-based AAV strategy reduces alcohol drinking in male, but not female, mice. This manipulation did not impact avoidance behavior, except in a fasted novelty-suppressed feeding test, in which vGAT shRNA mice demonstrated increased latency to feed on a familiar high-value food reward, an effect driven by male mice. In contrast, vGAT shRNA female mice showed heightened sensitivity to thermal stimulation. These data show a role for GABA release from central amygdala neurotensin neurons in modulating consumption of rewarding substances in different motivational states.

Insula Dynorphin and Kappa Opioid Receptor Systems Regulate Alcohol Drinking in a Sex-Specific Manner in Mice.

Alcohol use disorder is complex and multifaceted, involving the coordination of multiple signaling systems across numerous brain regions. Previous work has indicated that both the insular cortex and dynorphin (DYN)/kappa opioid receptor (KOR) systems contribute to excessive alcohol use. More recently, we identified a microcircuit in the medial aspect of the insular cortex that signals through DYN/KOR. Here, we explored the role of insula DYN/KOR circuit components on alcohol intake in a long-term intermittent access (IA) procedure. Using a combination of conditional knock-out strategies and site-directed pharmacology, we discovered distinct and sex-specific roles for insula DYN and KOR in alcohol drinking and related behavior. Our findings show that insula DYN deletion blocked escalated consumption and decreased the overall intake of and preference for alcohol in male and female mice. This effect was specific to alcohol in male mice, as DYN deletion did not impact sucrose intake. Further, insula KOR antagonism reduced alcohol intake and preference during the early phase of IA in male mice only. Alcohol consumption was not affected by insula KOR knockout in either sex. In addition, we found that long-term IA decreased the intrinsic excitability of DYN and deep layer pyramidal neurons (DLPNs) in the insula of male mice. Excitatory synaptic transmission was also impacted by IA, as it drove an increase in excitatory synaptic drive in both DYN neurons and DLPNs. Combined, our findings suggest there is a dynamic interplay between excessive alcohol consumption and insula DYN/KOR microcircuitry.SIGNIFICANCE STATEMENT The insular cortex is a complex region that serves as an integratory hub for sensory inputs. In our previous work, we identified a microcircuit in the insula that signals through the kappa opioid receptor (KOR) and its endogenous ligand dynorphin (DYN). Both the insula and DYN/KOR systems have been implicated in excessive alcohol use and alcohol use disorder (AUD). Here, we use converging approaches to determine how insula DYN/KOR microcircuit components contribute to escalated alcohol consumption. Our findings show that insula DYN/KOR systems regulate distinct phases of alcohol consumption in a sex-specific manner, which may contribute to the progression to AUD.

Patterned Feeding of a Hyper-Palatable Food (Oreo Cookies) Reduces Alcohol Drinking in Rats.

While a bidirectional positive link between palatable food intake and alcohol drinking has been suggested, several rodents studies report reduced alcohol drinking following palatable diets exposure. These studies utilized purified rodents’ diets high in sugar/fat; however, the effects of hyper-palatable food (HPF) rich in fat and sugar on alcohol drinking remain unclear. Furthermore, neural substrates involved in HPF-mediated changes in alcohol consumption are poorly understood. Therefore, the present study evaluated the effects of patterned feeding of a hyper-palatable food (Oreo cookies) on alcohol drinking as well as dopamine (DA) and serotonin (5-HT) content in rat’s mesocorticolimbic (medial-prefrontal cortex, orbitofrontal cortex, amygdala, and nucleus accumbens) circuitry. Male Long Evans rats received 8-weeks of intermittent (Mon, Tue, Wed) Oreo cookies access, which induced a patterned feeding, in which rats in the Oreo group overconsumed calories on HPF days whereas underconsumption was observed on chow only (Thu, Fri) days. Following HPF exposure, alcohol consumption was evaluated while patterned feeding continued. Alcohol intake in the Oreo group was significantly lower as compared to the chow controls. However, alcohol intake in the Oreo group increased to the levels seen in the group receiving chow following the suspension of patterned HPF feeding. Finally, DA levels in the nucleus accumbens were significantly greater, whereas its metabolite (DOPAC) levels were lower in the Oreo group compared to the chow controls. Surprisingly, 5-HT levels remained unaltered in all tested brain areas. Together, these data suggest that HPF-associated increased DA availability and reduced DA turnover within mesocorticolimbic circuitry may regulate alcohol drinking following patterned HPF feeding.

Vesicular glutamate transporter 2-containing neurons of the centrally-projecting Edinger-Westphal nucleus regulate alcohol drinking and body temperature

Previous studies in rodents have repeatedly demonstrated that the centrally-projecting Edinger-Westphal nucleus (EWcp) is highly sensitive to alcohol and is also involved in regulating alcohol intake and body temperature. Historically, the EWcp has been known as the main site of Urocortin 1 (Ucn1) expression, a corticotropin-releasing factor-related peptide, in the brain. However, the EWcp also contains other populations of neurons, including neurons that express the vesicular glutamate transporter 2 (Vglut2). Here we transduced the EWcp with adeno-associated viruses (AAVs) encoding Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the EWcp in alcohol drinking and in the regulation of body temperature. Activation of the EWcp with excitatory DREADDs inhibited alcohol intake in a 2-bottle choice procedure in male C57BL/6J mice, whereas inhibition of the EWcp with DREADDs had no effect. Surprisingly, analysis of DREADD expression indicated Ucn1-containing neurons of the EWcp did not express DREADDs. In contrast, AAVs transduced non-Ucn1-containing EWcp neurons. Subsequent experiments showed that the inhibitory effect of EWcp activation on alcohol intake was also present in male Ucn1 KO mice, suggesting that a Ucn1-devoid population of EWcp regulates alcohol intake. A final set of chemogenetic experiments showed that activation of Vglut2-expressing EWcp neurons inhibited alcohol intake and induced hypothermia in male and female mice. These studies expand on previous literature by indicating that a glutamatergic, Ucn1-devoid subpopulation of the EWcp regulates alcohol consumption and body temperature.

108. Synaptic Mechanisms of BNST CRF Neuron Excitability Regulating Alcohol Drinking Behavior and Anxiety

108. Synaptic Mechanisms of BNST CRF Neuron Excitability Regulating Alcohol Drinking Behavior and Anxiety

Erratum: The Decreased Cyclic-AMP Dependent-Protein Kinase A Function in the Nucleus Accumbens: A Role in Alcohol Drinking but not in Anxiety-Like Behaviors in Rats

The nucleus accumbens (NAc) brain structures have been implicated in the reward and reinforcing properties of ethanol. The present study investigated the role of nucleus accumbal cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling in alcohol drinking and anxiety-like behaviors of rats. It was found that infusion of PKA inhibitor (Rp-cAMP) into the NAc shell significantly increased the alcohol but not the sucrose intake, without modulating the anxiety-like behaviors, as measured by elevated plus maze test in rats. PKA inhibitor infusion into the NAc shell significantly decreased the protein levels of alpha-catalytic subunit of PKA (PKA-Calpha) and phosphorylated cAMP response element-binding protein (p-CREB) as well as decreased the protein levels of neuropeptide Y (NPY) in the shell but not in the NAc core of rats. On the other hand, infusion of PKA activator (Sp-cAMP) or NPY alone into the NAc shell did not produce any changes in alcohol intake; however, when these agents were coinfused with PKA inhibitor, they significantly attenuated the increases in alcohol preference induced by pharmacological inhibition of PKA. Interestingly, PKA activator coinfusion with PKA inhibitor into the NAc shell significantly normalized the PKA inhibitor-induced decreases in the protein levels of PKA-Calpha and p-CREB as well as of NPY in the NAc shell of rats. Taken together, these results provide the first evidence that decreased PKA function in the NAc shell is involved in alcohol drinking but not in anxiety-like behaviors of rats. Furthermore, decreased function of PKA may regulate alcohol drinking behaviors via CREB-mediated decreased expression of NPY in the NAc shell of rats.

The regulation of alcohol intake by melanin-concentrating hormone in rats

Given into the brain, melanin-concentrating hormone (MCH) increases alcohol consumption, but the mechanism and physiological relevance of this effect are unclear. We hypothesized that endogenous MCH will enhance alcohol drinking and that MCH increases alcohol's reinforcing properties. An MCH receptor 1 (MCHR1) antagonist, or saline was administered centrally alone, or preceding MCH or saline to rats trained to drink 10% alcohol using sucrose fading. Blocking MCHR1 neither reduced alcohol intake (saline = 0.4 ± 0.1 g, 30 μg MCHR1 antagonist = 0.4 ± 0.1 g/kg alcohol), nor attenuated MCH-induced alcohol drinking (MCHR1 antagonist/saline = 0.7 ± 0.1 g/kg, MCHR1 antagonist/MCH = 0.9 ± 0.1 g/kg alcohol). Another cohort of rats was trained to lever press for alcohol on a progressive ratio schedule. MCH or saline was administered centrally and lever presses were measured. MCH had no effect prior to the break point, but increased total responding during the session (saline = 87.2 ± 32.0, MCH = 315.4 ± 61.0 presses). In conclusion, these data suggest that MCH augments alcohol drinking partly by enhancing the drug's reinforcing value. Further, endogenous MCH does not seem to regulate alcohol drinking, however because the antagonist failed to attenuate MCH-induced alcohol intake this conclusion is tentative.

The Decreased Cyclic-AMP Dependent-Protein Kinase A Function in the Nucleus Accumbens: A Role in Alcohol Drinking but not in Anxiety-Like Behaviors in Rats

The nucleus accumbens (NAc) brain structures have been implicated in the reward and reinforcing properties of ethanol. The present study investigated the role of nucleus accumbal cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling in alcohol drinking and anxiety-like behaviors of rats. It was found that infusion of PKA inhibitor (Rp-cAMP) into the NAc shell significantly increased the alcohol but not the sucrose intake, without modulating the anxiety-like behaviors, as measured by elevated plus maze test in rats. PKA inhibitor infusion into the NAc shell significantly decreased the protein levels of alpha-catalytic subunit of PKA (PKA-Calpha) and phosphorylated cAMP response element-binding protein (p-CREB) as well as decreased the protein levels of neuropeptide Y (NPY) in the shell but not in the NAc core of rats. On the other hand, infusion of PKA activator (Sp-cAMP) or NPY alone into the NAc shell did not produce any changes in alcohol intake; however, when these agents were coinfused with PKA inhibitor, they significantly attenuated the increases in alcohol preference induced by pharmacological inhibition of PKA. Interestingly, PKA activator coinfusion with PKA inhibitor into the NAc shell significantly normalized the PKA inhibitor-induced decreases in the protein levels of PKA-Calpha and p-CREB as well as of NPY in the NAc shell of rats. Taken together, these results provide the first evidence that decreased PKA function in the NAc shell is involved in alcohol drinking but not in anxiety-like behaviors of rats. Furthermore, decreased function of PKA may regulate alcohol drinking behaviors via CREB-mediated decreased expression of NPY in the NAc shell of rats.

Regional heterogeneity for the intracranial self-administration of ethanol and acetaldehyde within the ventral tegmental area of alcohol-preferring (P) rats: involvement of dopamine and serotonin.

The meso-limbic dopamine (DA) system has an important role in regulating alcohol drinking. Previous findings from our laboratory indicated that Wistar rats self-administered ethanol (EtOH) directly into the posterior, but not anterior, ventral tegmental area (VTA), and that coadministration of a DA D(2,3) receptor agonist or a serotonin-3 (5-HT3) receptor antagonist blocked EtOH self-administration. In addition, we reported that alcohol-preferring (P) rats self-administered acetaldehyde (ACD), the first metabolite of EtOH, into the posterior VTA. The objectives of this study were to compare the reinforcing effects of EtOH and ACD within the VTA of P rats to examine the possibility that the reinforcing effects of EtOH within the VTA may be mediated by its conversion to ACD. Adult female P rats were stereotaxically implanted with guide cannulae aimed at either the posterior or anterior VTA. At 1 week after surgery, rats were placed in standard two-lever (active and inactive) experimental chambers for a total of seven to eight sessions. The 4-h sessions were conducted every other day. The results indicated that (a) 75-300 mg% (17-66 mM) EtOH and 6-90 microM ACD were self-administered into the posterior, but not anterior, VTA; (b) the self-administration of 150 mg% EtOH was not altered by coinfusion of a catalase inhibitor; (c) coadministration of the D(2/3) agonist quinpirole (100 microM) blocked the self-infusions of 150 mg% EtOH and 23 microM ACD into the posterior VTA; and (d) coadministration of 200 microM ICS205,930 (5-HT3 receptor antagonist) prevented the self-infusion of 150 mg% EtOH, whereas concentrations of ICS 205,930 up to 400 microM had no effect on the self-infusion of 23 microM ACD into the posterior VTA. Overall, the results of this study indicate that EtOH and ACD can independently produce reinforcing effects within the posterior VTA, and that activation of DA neurons mediates these effects. Furthermore, activation of 5-HT3 receptors within the posterior VTA is involved in the self-infusion of EtOH, but not ACD.

Serotonin-3 receptors in the actions of alcohol, alcohol reinforcement, and alcoholism.

This article represents the proceedings of a symposium at the 2003 annual meeting of the Research Society on Alcoholism in Fort Lauderdale, FL. The organizers and chairs were William J. McBride and David M. Lovinger. The presentations were (1) Mechanisms of alcohol potentiation of 5-HT3 receptor function, by David M. Lovinger and Tina Machu; (2) Chronic alcohol drinking alters 5-HT3 receptors regulating the mesolimbic dopamine system, by Richard J. Thielen; (3) 5-HT3 receptors in the VTA regulate alcohol drinking and the reinforcing effects of alcohol, by Zachary A. Rodd and James M. Murphy; and (4) Ondansetron as a treatment for "biological" alcoholism, by John D. Roache and Bankole A. Johnson.

Ethanol drinking and deprivation alter dopaminergic and serotonergic function in the nucleus accumbens of alcohol-preferring rats.

The alcohol deprivation effect is a temporary increase in the intake of, or preference for, ethanol after a period of deprivation that may result from persistent changes in key limbic regions thought to regulate alcohol drinking, such as the nucleus accumbens. The present study tested the hypothesis that chronic alcohol drinking under continuous 24-h free-choice conditions alters dopamine and serotonin neurotransmission in the nucleus accumbens and that these alterations persist in the absence of alcohol. Using the no-net-flux microdialysis method, the steady-state extracellular concentration (point of no-net-flux) for dopamine was approximately 25% higher in the adult female alcohol-preferring P rats given prior access to 10% ethanol, even after 2 weeks of ethanol abstinence, compared with the P rats gives access only to water. However, the extracellular concentration of serotonin was approximately 35% lower in animals given 8 weeks of continuous access to ethanol compared with water controls and animals deprived of ethanol for 2 weeks. The effect of local perfusion with 100 microM sulpiride (D(2) receptor antagonist) and 35 microM 1-(m-chlorophenyl)-biguanide (5-hydroxytryptamine(3) receptor agonist) on dopamine overflow were reduced approximately 33% in both groups of ethanol-exposed P rats compared with water controls. Free-choice alcohol drinking by P rats alters dopamine and serotonin neurotransmission in the nucleus accumbens, and many of these effects persist for at least 2 weeks in the absence of ethanol, suggesting that these underlying persistent changes may be in part responsible for increased ethanol drinking observed in the alcohol-deprivation effect.

Effects of 5-HT 3 receptor antagonists on daily alcohol intake under acquisition, maintenance, and relapse conditions in alcohol-preferring (P) rats

Previous research indicated that 5-HT 3 antagonists can reduce ethanol drinking in rats, but drinking conditions and other environmental manipulations influenced the efficacy of these antagonists. The current experiments were conducted to examine the effects of the 5-HT 3 antagonists MDL 72222 (MDL) or ICS 205-930 (ICS) on 24-h ethanol (10% v/v) consumption during acquisition, maintenance, and following a period of deprivation in selectively bred high alcohol-preferring (P) male rats. In an analysis of the acquisition of ethanol consumption, daily injections of MDL (1 mg/kg; s.c.) or ICS (1 or 5 mg/kg) were administered to separate groups of P rats during the initial 10 days of ethanol exposure. To examine the maintenance of ethanol drinking, these same groups of rats were allowed access to ethanol for 21 days with no pharmacological manipulations, and were then administered either saline or the 5-HT 3 antagonist. To examine the effects of a 5-HT 3 antagonist on relapse of ethanol drinking, another group of P rats was allowed access to ethanol for 6 weeks and was then deprived of ethanol for 3 weeks. Prior to ethanol reinstatement, rats were treated chronically (seven daily injections) or acutely with MDL (1 mg/kg), saline, or received no injections. MDL (1 mg/kg) and ICS (1 or 5 mg/kg) reduced ethanol intake during acquisition (60–80%) and during maintenance drinking (35–70%) in P rats pretreated with saline during acquisition. However, in rats pretreated with MDL or ICS during acquisition, there was a significant reduction in the effectiveness of either MDL or ICS to reduce ongoing ethanol drinking. Neither acute nor chronic treatment with 1 mg/kg MDL altered the 80% increase in ethanol consumption observed on the first day of reinstatement following a 3-week deprivation period. However, in a follow-up study, acute treatment with MDL (3 mg/kg) or ICS (5 mg/kg) did prevent the 80% increase in ethanol consumption observed on the first day of reinstatement. Overall, the results suggest that 5-HT 3 receptors are involved in the acquisition and maintenance of 24-h ethanol drinking, and that neuroadaptations may occur as a result of chronic treatment with 5-HT 3 antagonists, or during prolonged alcohol deprivation, which alter the involvement of these receptors in regulating alcohol drinking in the P rat.

Genetic factors which regulate alcohol drinking behavior and their effects on health status

High alcohol sensitivity common among Orientals is mainly due to genetic polymorphism in the low K(m) aldehyde dehydrogenase (ALDH2) gene. The relation of the ALDH2 genotype to alcohol sensitivity and drinking behavior was investigated in a Japanese occupational population. The frequency of alcohol-associated symptoms generally increased in the order of the typical homozygote, heterozygote, and atypical homozygote. Both drinking frequency and amounts of alcohol consumption were also significantly affected by the polymorphism. Polymorphism in the alcohol dehydrogenase beta-subunit (ADH2 gene) appeared to contribute to skin flushing post-alcohol exposure but not to alcohol drinking behavior. Multivariate analysis revealed that high alcohol consumption, the ALDH2*1/*1 genotype, and high daily hassles levels significantly contribute to the prevalence of those with a high problem-drinking score in an occupational population. In the study to assess the effects of the ALDH2 polymorphism and alcohol use on the induction of chromosome alterations in peripheral lymphocytes, we found that lymphocytes from habitual drinkers with the atypical ALDH2 genotypes had significantly higher frequencies of sister-chromatid exchange (SCE) than those from the typical ALDH2 genotype. We also measured acetaldehyde reversibly bound to hemoglobin (HbAA). In volunteers with the ALDH2*1/*2 genotype, the HbAA levels increased immediately after the drink and the elevated levels persisted up to 48 h. Among male workers, HbAA levels were significantly correlated with the recent alcohol consumption levels in both the ALDH2*1/*1 and ALDH2*1/*2 genotypes. However, the slope was much steeper in the ALDH2*1/*2 than in the ALDH2*1/*1. SCE and HbAA may be utilized as a good biomarker for health problems in the atypical ALDH2 genotype. Further extensive studies are required for evaluation of the interactive effects of genetic and environmental factors on alcohol-related health problems.

Alcohol Intake of P Rats Is Regulated by Muscarinic Receptors in the Pedunculopontine Nucleus and VTA

Experiments were conducted to determine whether muscarinic receptors within the pedunculopontine nucleus (PPN) and ventral tegmental area (VTA) are involved in regulating ethanol drinking behavior in the alcohol-preferring P line of rats. Female P rats were given limited access (2 h/day) to 10% (v/v) ethanol and 0.0125% (g/100 ml) saccharin solutions. Food was available ad libitum. Cholinergic agents were microinjected unilaterally into the PPN or VTA immediately prior to ethanol access. Intra-PPN carbachol (1–4 μg/0.5μl), which can inhibit cholinergic neuronal activity within the PPN, decreased ethanol (70% decrease at the highest dose; p < 0.05) and saccharin (90% decrease at the highest dose; p < 0.05) intake in a dose-dependent manner within the first 30 min. Intra-PPN scopolamine (5–15 μg/0.5 μl), which can stimulate cholinergic neuronal activity within the PPN, decreased ethanol intake in a dose-dependent manner within the first 30 min (65% decrease at the highest dose; p < 0.05) without reducing saccharin intake. Intra-VTA methylscopolamine (1–10 μg/0.5 μl), a muscarinic antagonist, significantly ( p < 0.05) reduced ethanol (60% decrease at the highest dose) and saccharin (50% decrease at the highest dose) intakes during the 2-h access period. Intra-VTA carbachol, a cholinergic agonist (1 and 2 μg/0.5 μl) decreased ethanol consumption in a dose-dependent manner within the first 60 min (50% decrease at the highest dose) without reducing saccharin intake. Overall, these results support an involvement of the cholinergic PPN–VTA system in regulating alcohol drinking and general consummatory behaviors of the P line of rats.

Involvement of CNS cholinergic systems in alcohol drinking of P rats.

Experiments were undertaken to determine if CNS muscarinic- and nicotinic-cholinergic receptors are involved in regulating alcohol drinking of rats from the selectively-bred alcohol-preferring P line. Intracerebroventricular (i.c.v.) drug infusions were administered into the lateral ventricle of female P rats 15 minutes before ethanol access. The muscarinic antagonists pirenzepine and scopolamine were tested on limited access (4 hours/day) to a 10% (v/v) ethanol solution. Food and water were available ad libitum. Nicotine and the nicotinic antagonist mecamylamine were tested on limited access (4 hours/day) to 10% (v/v) ethanol and 0.0125% saccharin solutions. Food was available ad libitum and water was available during the remaining 20 hours. The baseline ethanol intakes ranged between an average of 3.0 ± 0.3 g/kg/4 hours and 3.4 ± 0.3 g/kg/4 hours. Administration of 40-100 m g pirenzepine (M1-selective antagonist) had no effect on ethanol, food or water consumption. However, 20-80 m g scopolamine, a non-selective muscarinic antagonist, dosedependently decreased ethanol intake as much as 60% (p < 0.05) without altering food or water consumption. The nicotinic antagonist mecamylamine (20-120 m g) did not alter ethanol intake, but nicotine (40-80 m g) dose-dependently decreased ethanol drinking as much as 60% within the first 30 minutes (p < 0.05) without an effect on saccharin intake. The results suggest that: (a), muscarinic receptors, with the possible exception of the M1 subtype, are involved in regulating alcohol drinking and (b), activation of nicotinic receptors can reduce alcohol drinking of the P line of rats.

Serotonin, dopamine and GABA involvement in alcohol drinking of selectively bred rats

Neurochemical and neuropharmacological studies were undertaken to assess the involvement of CNS serotonin (5-HT), dopamine (DA) and GABA systems in regulating the alcohol-drinking behavior of two lines of rats selectively bred for their high alcohol-seeking behavior, namely the alcohol-preferring P line and the high alcohol-drinking HAD line of rats. Neurochemical data indicate that high alcohol-seeking behavior (when compared with data from rats with low alcohol-seeking characteristics) is associated with: a) lower (10–20%; p<0.05) contents of 5-HT in certain limbic regions (e.g., nucleus accumbens, frontal cortex, hypothalamus and hippocampus); b) a lower (10–15%; p<0.05) content of DA in the nucleus accumbens; c) higher (20–35%; p<0.05) densities of 5-HT 1A binding sites in some limbic regions (e.g., medial nucleus accumbens, medial prefrontal cortex and ventral hippocampus); and d) a greater (20–50%) density of GABA axon terminals in the nucleus accumbens. Furthermore, the acute administration of high doses of ethanol appears to increase the activity of the 5-HT and DA projections to the nucleus accumbens of the P line of rats (as indicated by the 20–30% elevated tissue levels of 5-HT and DA metabolites following IP ethanol administration); neuronal tolerance to alcohol appears to develop in both these monoamine pathways, as suggested by an attenuated effect on metabolite levels by a challenge dose of ethanol given to P rats that had been chronically drinking alcohol. The IP administration of agents which can increase the physiologically active pool of 5-HT (e.g., fluoxetine, an uptake inhibitor; fenfluramine, a releaser; and D,L-5-hydroxytryptophan, an immediate precursor) or which can mimic 5-HT (e.g., 5-HT 1 and 5-HT 2 agonists) all significantly decreased the volitional alcohol intake of the high alcohol-seeking rats. Similarly, the IP administration of a DA uptake inhibitor, DA releaser or D 2 agonist also reduced the volitional oral intake of alcohol by the P line of rats. In addition, the consumption of alcohol by the P line of rats is reduced by IP administration of Ro 15-4513, an inverse agonist at the GABA A-benzodiazepine-Cl − receptor complex. Overall, the data suggest that abnormalities exist in certain 5-HT, DA and GABA systems in the CNS of P and HAD rats and that these abnormal transmitter systems may be major underlying biological factors contributing to their high alcohol-seeking characteristics. A hypothesis is offered to explain the involvement of the 5-HT, DA and GABA systems of the nucleus accumbens in regulating alcohol drinking of the selectively bred P and HAD lines of rats.