Early life stress is a risk factor for excessive alcohol drinking and impulsivity in adults and is mediated via a CRF/GABA(A) mechanism.

Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2ß3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the development of impulsivity and excessive alcohol consumption.

CRF-amplified neuronal TLR4/MCP-1 signaling regulates alcohol self-administration.

Alcohol dependence is a complex disorder that initiates with episodes of excessive alcohol drinking known as binge drinking. It has a 50-60% risk contribution from inherited susceptibility genes; however, their exact identity and function are still poorly understood. We report that alcohol-preferring P rats have innately elevated levels of Toll-like receptor 4 (TLR4) and monocyte chemotactic protein-1 (MCP-1) that colocalize in neurons from the central nucleus of the amygdala (CeA) and ventral tegmental area (VTA). To examine the potential role of a TLR4/MCP-1 signal, we used Herpes Simplex Virus (HSV) vectors (amplicons) that retain in vivo neurotropism. Infusion of amplicons for TLR4 or MCP-1 siRNA into the CeA or VTA from the P rats inhibited target gene expression and blunted binge drinking. A similarly delivered amplicon for scrambled siRNA did not inhibit TLR4 or MCP-1 expression nor reduce binge drinking, identifying a neuronal TLR4/MCP-1 signal that regulates the initiation of voluntary alcohol self-administration. The signal was sustained during alcohol drinking by increased expression of corticotropin-releasing factor and its feedback regulation of TLR4 expression, likely contributing to the transition to alcohol dependence.

Triple monoamine uptake inhibitors demonstrate a pharmacologic association between excessive drinking and impulsivity in high-alcohol-preferring (HAP) mice.

Approximately 30% of current drinkers in the United States drink excessively, and are referred to as problem/hazardous drinkers. These individuals, who may not meet criteria for alcohol abuse or dependence, comprise binge, heavy drinkers, or both. Given their high prevalence, interventions that reduce the risk of binge and heavy drinking have important public health implications. Impulsivity has been repeatedly associated with excessive drinking in the clinical literature. As impulsivity is correlated with, and may play a critical role in, the initiation and maintenance of excessive drinking, this behavior may be an important target for therapeutic intervention. Hence, a better understanding of pharmacological treatments capable of attenuating excessive drinking and impulsivity may markedly improve clinical outcomes. The high-alcohol-preferring (HAP) mice represent a strong rodent model to study the relationship between impulsivity and excessive alcohol drinking, as recent evidence indicates they consume high levels of alcohol throughout their active cycle and are innately impulsive. Using this model, the present study demonstrates that the triple monoamine uptake inhibitors (TUIs) amitifadine and DOV 102, 677 effectively attenuate binge drinking, heavy drinking assessed via a 24-hour free-choice assay, and impulsivity measured by the delay discounting procedure. In contrast, 3-PBC, a GABA-A α1 preferring ligand with mixed agonist-antagonist properties, attenuates excessive drinking without affecting impulsivity. These findings suggest that in HAP mice, monoamine pathways may predominate as a common mechanism underlying impulsivity and excessive drinking, while the GABAergic system may be more salient in regulating excessive drinking. We further propose that TUIs such as amitifadine and DOV 102, 677 may be used to treat the co-occurrence of impulsivity and excessive drinking.

Amitifadine, a triple monoamine uptake inhibitor, reduces binge drinking and negative affect in an animal model of co-occurring alcoholism and depression symptomatology.

The co-occurrence of alcoholism and depression is highly prevalent and difficult to treat. In an animal model of binge drinking that exhibits abstinence-induced behaviors reminiscent of negative affective states, the triple monoamine uptake inhibitor, amitifadine, produced a selective, dose dependent attenuation of binge drinking. Amitifadine also reversed abstinence-induced increases in the intracranial self-stimulation threshold, a model of anhedonia, and immobility in the forced swim test, reflecting behavioral despair. In view of the safety profile of amitifadine in humans, including low risk for weight gain, lack of sexual side effects, and low potential for abuse, we hypothesize that amitifadine will be effective in treating co-occurring alcoholism and depression.

Effects of the triple monoamine uptake inhibitor DOV 102,677 on alcohol-motivated responding and antidepressant activity in alcohol-preferring (P) rats.

BACKGROUND: Concurrent inhibitors of dopamine, norepinephrine, and serotonin uptake have been proposed as novel antidepressants. Given the high comorbidity between alcoholism and depression, we evaluated the activity of DOV 102,677 (DOV) on alcohol-maintained responding and performance in the forced swim test (FST), a model of antidepressant (AD) activity, using alcohol-preferring (P) rats. METHODS: Following training to lever press for either alcohol (10% v/v) or sucrose (3, 2%, w/v) on a fixed-ratio 4 (FR4) schedule, DOV (1.56 to 50 mg/kg; PO) was given 25 minutes or 24 hours prior to evaluation. The effects of DOV (12.5 to 50 mg/kg; PO) in the FST were evaluated 25 minutes posttreatment. RESULTS: DOV (6.25 to 50 mg/kg) dose-dependently reduced alcohol-maintained responding by 59 to 88% at 25 minutes posttreatment, without significantly altering sucrose responding. The reduction in alcohol responding (44% at 50 mg/kg) was sustained for up to 120 hours after a single dose. Administration of a single dose of DOV (25, 50 mg/kg) 24 hours before testing suppressed alcohol responding for 48 hours by 59 to 62%. DOV (12.5 to 50 mg/kg) also dose-dependently reduced immobility of P rats in the FST. CONCLUSIONS: DOV produces both prolonged and selective reductions of alcohol-motivated behaviors in P rats. The elimination kinetics of DOV suggests that its long duration of action may be due to an active metabolite. DOV also produced robust AD-like effects in P rats. We propose that DOV may be useful in treating comorbid alcoholism and depression in humans.

Binge Drinking: In Search of its Molecular Target via the GABA(A) Receptor.

Binge drinking, frequently referred to clinically as problem or hazardous drinking, is a pattern of excessive alcohol intake characterized by blood alcohol levels ≥0.08 g% within a 2-h period. Here, we show that overexpression of α1 subunits of the GABA(A) receptor contributes to binge drinking, and further document that this involvement is related to the neuroanatomical localization of α1 receptor subunits. Using a herpes simplex virus amplicon vector to deliver small interference RNA (siRNA), we showed that siRNA specific for the α1 subunit (pHSVsiLA1) caused profound, long-term, and selective reduction of gene expression, receptor density, and binge drinking in high-alcohol drinking rats when delivered into the ventral pallidum (VP). Scrambled siRNA (pHSVsiNC) delivered similarly into the VP failed to alter gene expression, receptor density, or binge drinking. Silencing of the α1 gene in the VP, however, failed to alter binge sucrose or water intake. These results, along with our prior research, provide compelling evidence that the α1-containing GABA(A) receptor subunits are critical in the regulation of binge-like patterns of excessive drinking. Collectively, these data may be useful in the development of gene-based and novel pharmacological approaches for the treatment of excessive drinking.

α4-Containing GABA(A) Receptors are Required for Antagonism of Ethanol-Induced Motor Incoordination and Hypnosis by the Imidazobenzodiazepine Ro15-4513.

Alcohol (ethanol) is widely consumed for its desirable effects but unfortunately has strong addiction potential. Some imidazobenzodiazepines such as Ro15-4513 are able to antagonize many ethanol-induced behaviors. Controversial biochemical and pharmacological evidence suggest that the effects of these ethanol antagonists and ethanol are mediated specifically via overlapping binding sites on α4/δ-containing GABA(A)-Rs. To investigate the requirement of α4-containing GABA(A)-Rs in the mechanism of action of Ro15-4513 on behavior, wildtype (WT) and α4 knockout (KO) mice were compared for antagonism of ethanol-induced motor incoordination and hypnosis. Motor effects of ethanol were tested in two different fixed speed rotarod assays. In the first experiment, mice were injected with 2.0 g/kg ethanol followed 5 min later by 10 mg/kg Ro15-4513 (or vehicle) and tested on a rotarod at 8 rpm. In the second experiment, mice received a single injection of 1.5 g/kg ethanol ± 3 mg/kg Ro15-4513 and were tested on a rotarod at 12 rpm. In both experiments, the robust Ro15-4513 antagonism of ethanol-induced motor ataxia that was observed in WT mice was absent in KO mice. A loss of righting reflex (LORR) assay was used to test Ro15-4513 (20 mg/kg) antagonism of ethanol (3.5 g/kg)-induced hypnosis. An effect of sex was observed on the LORR assay, so males and females were analyzed separately. In male mice, Ro15-4513 markedly reduced ethanol-induced LORR in WT controls, but α4 KO mice were insensitive to this effect of Ro15-4513. In contrast, female KO mice did not differ from WT controls in the antagonistic effects of Ro15-4513 on ethanol-induced LORR. We conclude that Ro15-4513 requires α4-containing receptors for antagonism of ethanol-induced LORR (in males) and motor ataxia.

Binge alcohol drinking is associated with GABAA alpha2-regulated Toll-like receptor 4 (TLR4) expression in the central amygdala.

Binge drinking (blood-alcohol levels ≥ 0.08 g% in a 2-h period), is a significant public health burden in need of improved treatment. Gene therapy may offer beneficial alternatives to current psychosocial and pharmacotherapeutic interventions, but identification of the target genes is a clinical challenge. We report that a GABA(A) α2 siRNA vector (pHSVsiLA2) infused into the central nucleus of the amygdala (CeA) of alcohol-preferring (P) rats caused profound and selective reduction of binge drinking associated with inhibition of α2 expression, decreased GABA(A) receptor density, and inhibition of Toll-like receptor 4 (TLR4). CeA infusion of a TLR4 siRNA vector (pHSVsiLTLR4a) also inhibited binge drinking, but neither vector functioned when infused into the ventral pallidum. Binge drinking was inhibited by a GABA(A) α1 siRNA vector (pHSVsiLA1) infused into the ventral pallidum, unrelated to TLR4. The vectors did not alter sucrose intake and a scrambled siRNA vector was negative. The data indicate that GABA(A) α2-regulated TLR4 expression in the CeA contributes to binge drinking and may be a key early neuroadaptation in excessive drinking.

Operant self-administration models for testing the neuropharmacological basis of ethanol consumption in rats.

Operant self-administration procedures are used to assess the neural basis of ethanol-seeking behavior under a wide range of experimental conditions. In general, rats do not spontaneously self-administer ethanol in pharmacologically meaningful amounts. This unit provides a step-by-step guide for training rats to self-administer quantities of ethanol that produce moderate to high blood-alcohol content. Different protocols are used for rats that are genetically heterogeneous versus rats that are selectively bred for high alcohol preference. Also, these protocols have different sets of advantages and disadvantages in terms of the ability to control for caloric intake and taste of solutions in operant testing. Basic self-administration protocols can also be altered to focus on different aspects of the motivational properties of ethanol (for example, those related to dependence). This unit provides multiple protocols that lead to alcohol intake in rats, which can be pharmacologically probed relative to a variety of control conditions.

Vitamin A deficiency and behavioral and motor deficits in the human immunodeficiency virus type 1 transgenic rat.

The human immunodeficiency virus type 1 (HIV-1) transgenic (Tg) rat model incorporates a noninfectious viral genome that is under similar regulatory control mechanisms in vivo as those that exist with natural infection in humans. Vitamin A (VA) deficiency in humans has been associated with progressive systemic HIV disease and with impaired cognition in rodent models. The effects on of VA deficiency on the development of behavioral abnormalities with HIV infection have not been previously described. In these studies, wild-type (Wt) and Tg rats maintained on either a normal (VA+) or a VA-deficient (VA-) diet were examined for activity in an open field (horizontal activity, total distance, vertical activity, and rearing) and on rotarod testing. On both open field and rotarod testing, the Tg rats performed worse than the Wt rats, with the most severe deficits noted in the TgVA- animals. Analysis of the specific effects of the presence of the HIV transgene and the diet on the performance on the open field tests showed a dominant effect from the transgene on all of the tests, with an effect from the diet on only the number of rearings. On rotarod testing, effects form both the diet and the transgene were observed at lower speeds, at the highest speeds, and on the accelerating rotarod. These studies therefore demonstrate that behavioral and motor abnormalities can be detected in this model and are likely due to similar mechanisms by which humans infected with HIV might develop cognitive-motor impairment in association with VA deficiency.

The alpha1 subunit of the GABA(A) receptor modulates fear learning and plasticity in the lateral amygdala.

Synaptic plasticity in the amygdala is essential for emotional learning. Fear conditioning, for example, depends on changes in excitatory transmission that occur following NMDA receptor activation and AMPA receptor modification in this region. The role of these and other glutamatergic mechanisms have been studied extensively in this circuit while relatively little is known about the contribution of inhibitory transmission. The current experiments addressed this issue by examining the role of the GABA(A) receptor subunit alpha1 in fear learning and plasticity. We first confirmed previous findings that the alpha1 subunit is highly expressed in the lateral nucleus of the amygdala. Consistent with this observation, genetic deletion of this subunit selectively enhanced plasticity in the lateral amygdala and increased auditory fear conditioning. Mice with selective deletion of alpha1 in excitatory cells did not exhibit enhanced learning. Finally, infusion of a alpha1 receptor antagonist into the lateral amygdala selectively impaired auditory fear learning. Together, these results suggest that inhibitory transmission mediated by alpha1-containing GABA(A) receptors plays a critical role in amygdala plasticity and fear learning.

Deficits in substance P mRNA levels in the CeA are inversely associated with alcohol-motivated responding.

In the present study, in vitro and in vivo studies were conducted to determine the relationship between innate substance P (SP) levels and alcohol-motivated behavior in alcohol-preferring (P) and nonpreferring (NP) rat lines. In Experiment 1, in situ hybridization and quantitative autoradiography were used to detect and measure SP mRNA levels in discrete brain loci of the P and NP rats. The results indicated significantly lower SP mRNA levels in the central nucleus of the amygdala (CeA) of P compared with those of NP rats. Experiment 2 evaluated the effects of SP, microinfused into the CeA, on alcohol (10%, v/v) and sucrose (2%, w/v) motivated responding in the P rat. The results revealed that, when infused into the CeA (1-8 microg), SP reduced alcohol responding by 48-85% of control levels, with no effects on sucrose responding. Neuroanatomical control infusions (1-8 microg) into the caudate putamen (CPu) also failed to significantly alter alcohol- or sucrose-motivated behaviors. Given the selective reductions on alcohol (compared to sucrose) responding by direct intracranial infusion of SP, the data suggest that deficits in SP signaling within the CeA (an anxiety regulating locus) are inversely associated with alcohol-motivated behaviors. Activation of SP receptors in the CeA may reduce anxiety-like behavior in the P rat and contribute to reductions on alcohol responding. The SP system may be a suitable target for the development of drugs to reduce alcohol-drinking behavior in humans.

Responding for brain stimulation reward in the bed nucleus of the stria terminalis in alcohol-preferring rats following alcohol and amphetamine pretreatments.

The bed nucleus of the stria terminalis (BNST) has been reported to release increased levels of extracellular dopamine (DA) following the systemic administration of abused drugs in outbred rats. This study examined the BNST as a novel locus for supporting operant responding for brain stimulation reward (BSR) in rats bred for alcohol preference while determining any potentiating effects of ethanol (EtOH) (0.125-1.25 g/kg, i.p.) and amphetamine (0.25-1.60 mg/kg, i.p.) on BSR within the BNST. Also examined was the capability of D1 receptor blockade to attenuate any observed potentiation. Following surgical implantation, alcohol-preferring (P) and non-preferring (NP) rats responded to a range of descending frequencies (300-20 Hz) as evaluated by a rate-frequency paradigm. The results revealed that the BNST was capable of supporting BSR in P but not NP rats. Also, amphetamine pretreatment produced a significant leftward shift in the rate-frequency function in P rats with significant reductions observed in three other measures of reward threshold, while EtOH only lowered the minimum frequency needed to produce responding. The effects of systemic amphetamine were successfully attenuated by the unilateral infusion of the D1 receptor antagonist SCH 23390 (5.0 microg) into the contralateral nucleus accumbens. The results suggest the BNST is capable of supporting BSR performance in P, but not NP rats, possibly due to increased sensitivity to the electrical stimulation-induced DA release of BSR in the innately DA "deficient" limbic system of P rats.

Blockade of GABA(A) receptors within the extended amygdala attenuates D(2) regulation of alcohol-motivated behaviors in the ventral tegmental area of alcohol-preferring (P) rats.

The dopamine (DA) mesolimbic pathway, which originates from DA cell bodies within the ventral tegmental area (VTA), has been shown by various studies to play a role in the mediation of various drugs of abuse including alcohol (EtOH). It has been suggested that the VTA's control of EtOH reward is mediated in part by the D2 receptors within the VTA. These receptors may be under the regulation of reciprocal GABAergic inputs from forebrain components of the mesolimbic path such as the nucleus accumbens (NAcc), a classic EtOH reward substrate, and the bed nucleus of the stria terminalis, a substrate recently implicated in EtOH reinforcement, forming a self-regulating feedback loop. To test this hypothesis, D2 regulation of EtOH self-administration (SA) was evaluated by the microinfusion of the D2 antagonist eticlopride into the VTA of P rats, which produced profound reductions in EtOH SA in the highest (20.0 and 40.0microg) doses tested in both BST/VTA and NAcc/VTA implanted P rats. To determine the role of GABA in the mediation of EtOH SA, a 32.0ng dose the non-selective GABA antagonist SR 95531 was microinfused into the BST producing no effect on responding for EtOH and into the NAcc which lead to a reduction in EtOH responding. Finally, the hypothesis that GABA innervation of the VTA from the mesolimbic forebrain may influence EtOH SA was examined by the simultaneous infusion of eticlopride (40.0microg) into the VTA and SR 95531 (32.0ng) into either the BST or NAcc. This combination infusion completely attenuated the reduction in EtOH SA observed with the 40.0microg dose of eticlopride alone in both groups of animals. These results suggest that while the D2 receptors within the VTA regulate EtOH-motivated behaviors, this is modulated by GABAergic input from the mesolimbic forebrain, specifically from the BST and NAcc.

Dopamine and benzodiazepine-dependent mechanisms regulate the EtOH-enhanced locomotor stimulation in the GABAA alpha1 subunit null mutant mice.

The present study investigated the role of the alpha1-containing GABA(A) receptors in the neurobehavioral actions of alcohol. In Experiment 1, mice lacking the alpha1 subunit (alpha1 (-/-)) were tested for their capacity to initiate operant-lever press responding for alcohol or sucrose. Alcohol intake in the home cage was also measured. In Experiment 2, the alpha1 (-/-) mice were injected with a range of alcohol doses (0.875-4.0 g/kg; i.p.) to evaluate the significance of the alpha1 subunit in alcohol's stimulant actions. In Experiment 3, we determined if the alcohol-induced stimulant effects were regulated via dopaminergic (DA) or benzodiazepine (BDZ)-dependent mechanisms. To accomplish this, we investigated the capacity of DA (eticlopride, SCH 23390) and BDZ (flumazenil, betaCCt) receptor antagonists to attenuate the alcohol-induced stimulant actions. Compared with wild-type mice (alpha1 (+/+)), the null mutants showed marked reductions in both EtOH and sucrose-maintained responding, and home-cage alcohol drinking. The null mutants also showed significant increases in locomotor behaviors after injections of low-moderate alcohol doses (1.75-3.0 g/kg). betaCCt, flumazenil, eticlopride, and SCH 23390 were able to attenuate the alcohol-induced stimulation in mutant mice, in the absence of intrinsic effects. These data suggest the alpha1 receptor plays an important role in alcohol-motivated behaviors; however, it also appears crucial in regulating the reinforcing properties associated with normal ingestive behaviors. Deleting the alpha1 subunit of the GABA(A) receptor appears to unmask alcohol's stimulatory effects; these effects appear to be regulated via an interaction of both DA- and GABA(A) BDZ-dependent mechanisms.

Decreased reward during acute alcohol withdrawal in rats selectively bred for low alcohol drinking.

We have previously hypothesized that increased sensitivity to the dysphoric-like or aversive effects of alcohol withdrawal following an initial exposure to alcohol might be associated with a genetic propensity to avoid alcohol. A decrease in brain reward function, as measured by an elevation in intracranial self-stimulation (ICSS) reward threshold, is one of the few methods available to model dysphoric-like or aversive effects of drug withdrawal in rats. We compared brain reward function during withdrawal following an initial exposure to alcohol in alcohol-naïve rats selectively bred for high (HAD1 line) versus low (LAD1 line) voluntary alcohol consumption. Male HAD1 (n=5) and LAD1 (n=6) rats were implanted with unilateral electrodes in the medial forebrain bundle and trained to bar press for delivery of a 100 microA current that varied in frequency from 45 to 200 Hz. Responding for ICSS was generally stable within subjects across multiple experimental sessions on a given day and across several consecutive days prior to alcohol or water administration. ICSS responding was assessed in both rat lines prior to and at 12, 14, 16, 18, 20, and 24 h following a single intragastric infusion of alcohol (4.0 g/kg body weight) or water. Rats of the LAD1 line, but not those of the HAD1 line, exhibited a decrease in brain reward function as evidenced by a decrease in bar-press responding for ICSS and an increase in ICSS stimulation threshold during alcohol withdrawal. The results suggest that rats selectively bred for low alcohol drinking may experience dysphoric-like effects during withdrawal from an initial exposure to alcohol, while rats selectively bred for high alcohol drinking may not.

Amphetamine lowers brain stimulation reward (BSR) threshold in alcohol-preferring (P) and -nonpreferring (NP) rats: regulation by D-sub-1 and D-sub-2 receptors in the nucleus accumbens.

Differences in the mesolimbic dopamine (DA) pathway that regulates alcohol preference may also increase sensitivity to the reinforcing effects of other drugs of abuse. In the present study, the curve-shift (rate-frequency) paradigm was used to quantify the interaction of amphetamine with the rewarding effects of lateral hypothalamic brain stimulation reward (BSR) in alcohol-preferring (P) and -nonpreferring (NP) rats. The role of D-sub-1 and D-sub-2 DA receptors of the nucleus accumbens (NAcc) in mediating the reward-potentiating effects of amphetamine was also determined. Animals were tested with randomly administered amphetamine (0.25, 0.75, 1.25 mg/kg ip), DA-receptor antagonists (SCH 23390 [2.0 microg, 5.0 microg]; eticlopride [2.0 microg, 5.0 microg]), or a combination of the 2 (SCH 23390 [2.0 microg, 5.0 microg] + 0.75 mg/kg amphetamine; eticlopride [2.0 microg, 5.0 microg] + 0.75 mg/kg amphetamine). Amphetamine produced comparable dose-related leftward shifts in the rate-frequency function for both P and NP rats, with a greater than 60% reduction observed in BSR threshold. On intervening days, baseline threshold was unaltered between tests and similar between rat lines. Unilateral infusion in the NAcc of either the D-sub-1 or D-sub-2 receptor antagonist produced rightward shifts in the rate-frequency function of amphetamine, completely reversing-attenuating its reward-enhancing effects. The results demonstrate that amphetamine produces similar threshold-lowering effects in both P and NP rats and that the reward-potentiating effects of amphetamine do not correlate with alcohol preference under the conditions of the present study.

Selective GABAA alpha5 benzodiazepine inverse agonist antagonizes the neurobehavioral actions of alcohol.

BACKGROUND: Previous research has implicated the alpha5-containing GABAA receptors of the hippocampus in the reinforcing properties of alcohol. In the present study, a selective GABAA alpha5 benzodiazepine inverse agonist (e.g., RY 023) was used in a series of in vivo and in vitro studies to determine the significance of the alpha5-receptor in the neurobehavioral actions of alcohol. METHODS: In experiment one, systemic injections of RY 023 (1 to 10 mg/kg IP) dose-dependently reduced ethanol-maintained responding by 52% to 86% of controls, whereas bilateral hippocampal infusions (0.3 to 20 microg) reduced responding by 66% to 84% of controls. Saccharin responding was reduced only with the highest intraperitoneal (e.g., 10 mg) and microinjected (e.g., 20 microg) doses. In experiment two, RY 023 (3.0 to 15 mg/kg IP) reversed the motor-impairing effects of a moderate dose of alcohol (0.75 g/kg) on an oscillating bar task in the absence of intrinsic effects. In the open field, RY 023 (3.0 to 7.5 mg/kg) produced intrinsic effects alone but attenuated the suppression of the 1.25 g/kg ethanol dose. Because the diazepam-insensitive receptors (e.g., alpha4 and alpha6) have been suggested to play a role in alcohol motor impairing and sedative actions, experiment three compared the efficacy of RY 023 with Ro 15-4513 and two prototypical benzodiazepine antagonists (e.g., flumazenil and ZK 93426) across the alpha4beta3gamma2-, alpha5beta3gamma2-, and alpha6beta3gamma2-receptor subtypes in Xenopus oocytes. RESULTS: RY 023 produced classic inverse agonism at all receptor subtypes, whereas Ro15-4513 and the two antagonists displayed a neutral or agonistic profile at the diazepam-insensitive receptors. CONCLUSIONS: Overall, the results extend our previous findings by demonstrating that an alpha5-subtype ligand is capable of attenuating not only the rewarding action of alcohol but also its motor impairing and sedative effects. We propose that these actions are mediated in part by the alpha5-receptors of the hippocampus. The hippocampal alpha5-receptors could represent novel targets in understanding the neuromechanisms regulating the neurobehavioral actions of alcohol in humans.

Brain stimulation reward performance and sucrose maintained behaviors in alcohol-preferring and -nonpreferring rats.

BACKGROUND: The relation between ethanol (EtOH) preference and sensitivity to brain stimulation reward (BSR) was examined under multiples schedules of reinforcement in the current study. For comparison, the study also examined the relation between EtOH preference and motivation for a sweet, palatable sucrose solution under similar schedules of reinforcement. METHODS: To investigate sensitivity to BSR performance, alcohol-preferring and -nonpreferring rats were tested using the curve-shift (rate-frequency) paradigm under several intensity levels during a 20-min session. Animals were first trained under an optimal current intensity, which produced maximal responding (i.e., 100%) across a series of descending frequencies (i.e., 300-20 Hz). BSR was then evaluated at 100%, 75%, and 50% of the optimal current. The sensitivity of the curve-shift method was further evaluated under the animal's optimal current using the FR1, FR6, and FR12 schedules. To examine responding for the sucrose solution, a separate group of alcohol-preferring and -nonpreferring rats was initially stabilized on an FR1 schedule and then subsequently on FR6 and FR12 schedules. RESULTS: The results demonstrated that reducing the reinforcing efficacy of BSR via reduction in current intensity/reinforcement schedule produced marked orderly rightward shifts in the rate-frequency curves relating responding to stimulation frequency in both rat lines. However, no differences were found between the lines with either manipulation. Specifically, both lines demonstrated orderly reductions in response rate and increases in BSR threshold parameters (i.e., half maximal frequency/responding, minimum and maximum frequencies). In contrast to BSR, genetic selection for EtOH preference was highly associated with responding for the sweet, palatable sucrose solution. The association was even more salient as the reinforcement schedule increased (i.e., reward cost). CONCLUSION: The results demonstrate that responding for BSR is not associated with EtOH preference, insofar as alcohol-preferring and -nonpreferring rats respond similarly under an array of reinforcement schedules and current intensities. In contrast, genetic selection for EtOH preference is highly associated with responding for a palatable sucrose reward, and the relation increases as the reward cost for the sucrose increases. These findings suggest that similar/overlapping mechanisms of action regulate the reinforcing properties of EtOH and sucrose but that overlapping yet distinct neuronal mechanism may modulate the reward characteristics of BSR and EtOH preference.

Central opioid receptors differentially regulate the nalmefene-induced suppression of ethanol- and saccharin-reinforced behaviors in alcohol-preferring (P) rats.

The exact opioid-sensitive receptors participating in EtOH-seeking behaviors remains unclear. Previous studies have reported higher densities of micro-opioid receptor binding in the nucleus accumbens (NACC) of P relative to NP rats; however, no differences were seen in delta-receptor binding. In contrast to the NACC, substantially lower levels of micro-receptor binding have been observed in the ventral tegmental area (VTA) of both P and NP rats, albeit no line differences have been observed. In the present study, opioid receptors in the NACC, VTA, and hippocampus were evaluated for their capacity to regulate both EtOH- and saccharin-motivated behaviors in the genetically selected alcohol-preferring (P) rat. To accomplish this, nalmefene, an opiate antagonist with preferential binding affinity for the micro-opioid receptor was unilaterally or bilaterally infused during concurrent availability of 1 h daily EtOH (10% v/v) and saccharin (0.025 or 0.050% w/v) solutions. Rats performed under a two-lever fixed ratio (FR) schedule in which four responses on one lever produced the EtOH solution, and four on a second lever produced the saccharin solution. The results demonstrated that when responding maintained by both EtOH and saccharin are matched at basal levels, unilateral (1-60 microg) or bilateral (0.5-10 microg) microinjections of nalmefene into the NACC produced selective dose-dependent reductions on responding maintained by EtOH. Unilateral (40, 60 microg) and bilateral (10 microg) VTA infusions were also observed to selectively reduced EtOH responding; however, greater nalmefene doses were required and the magnitude of suppression on EtOH responding was markedly less compared with the NACC. The greater sensitivity of nalmefene to suppress EtOH responding in the NACC is likely due to the greater number of opioid receptors in the NACC relative to the VTA. Only bilateral infusion of the 40 microg dose in the NACC and VTA suppressed responding maintained by both EtOH and saccharin. In contrast, intrahippocampal infusions dose dependently suppressed EtOH- and saccharin-maintained responding over a range of doses (1-20 microg). The present study provides evidence that nalmefene suppresses EtOH-motivated behaviors via blockade of opioid receptors within the NACC and VTA, and under various dose conditions both reinforcer and neuroanatomical specificity can be observed.