Alcohol Preference In Mice Research Articles

RNA Editing of Serotonin 2C Receptor and Alcohol Intake.

Serotonin 2C receptor (5-HT2CR) belongs to the superfamily of seven transmembrane domain receptors coupled to G proteins (GPCR). It is broadly distributed in the CNS and its expression is relatively high in the limbic system including the amygdala, nucleus accumbens (NAc), hippocampus, and hypothalamus. Based on its expression patterns and numerous pharmacological studies, 5-HT2CR is thought to be involved in various brain functions including emotion, appetite, and motor behavior. Here, we review 5-HT2CR and its relationship with alcohol intake with a particular focus on the involvement of 5-HT2CR mRNA editing and its association with alcohol preference in mice. RNA editing is a post-transcriptional modification mechanism. In mammals, adenosine is converted to inosine by the deamination enzymes ADAR1 and ADAR2. 5-HT2CR is the only GPCR subjected to RNA editing within the coding region. It has five editing sites in exon 5 that encode the second intracellular loop. Consequently, three amino acids residues (I156, N158, and I160) of the unedited receptor (INI) may be altered to differently edited isoforms, resulting in a change of receptor activity such as 5-HT potency and G-protein coupling. 5-HT2CR in the NAc is involved in enhanced alcohol drinking after chronic alcohol exposure and alterations in 5-HT2CR mRNA editing is important in determining the alcohol preference using different strains of mice and genetically modified mice. RNA editing of this receptor may participate in the development of alcoholism.

Cross‐Sensitization to Binge Alcohol Intake Following Binge Intake of Palatable Diets

Obesity and alcoholism, two of the most common chronic conditions in the United States, may be behaviorally linked as binge intake of palatable diets, such as diets high in fat (HFD), and binge alcohol (EtOH) intake may utilize similar neurocircuitry. We hypothesized that binge intake of one modality (i.e. HFD) may sensitize the shared neurocircuitry to further activation by another modality (i.e. EtOH). Therefore, we combined a binge HFD intake model in mice where HFD access is limited to one 24hr session per week (Limited Access HFD: LAH) with a patterned EtOH access schedule that also promotes binge‐like intake in a two‐bottle choice model (Limited Access EtOH: LAE; 4hrs/day, 4days/wk). Control mice were given the same EtOH access schedule but were given continuous ad libitum access to HFD (60% calories from fat) or control Chow diet (10% calories from fat). Six‐week old male C57Bl/6J mice were weight matched, individually housed, and assigned to HFD+LAE, Chow+LAE, or LAH+LAE groups (n=10 per group). All groups of mice were given increasing EtOH concentrations over the course of the study (10% EtOH for three weeks, 15% EtOH for two weeks, and 20% EtOH for two weeks). Body mass was measured daily while EtOH/water intake and EtOH preference were assessed after every drinking session. Over the course of the study, HFD+LAE mice gained significantly more weight than Chow+LAE or LAH+LAE mice (p=0.001; one‐way ANOVA). While weight gain was predominantly linear in HFD+LAE and Chow+LAE mice, LAH+LAE mice showed weight cycling indicating a binge‐like eating pattern. LAH+LAE mice consumed significantly more EtOH than Chow+LAE or HFD+LAE mice (p=0.001, repeated measures two‐way ANOVA), suggesting that binge intake of HFD led to increased binge EtOH intake patterns in LAH mice. LAH+LAE and Chow+LAE mice showed higher preference towards EtOH than HFD+LAE mice (p=0.001, repeated measures two‐way ANOVA). Together, these results suggest limited access to palatable diets induce binge‐like eating patterns that further cross‐sensitize to binge alcohol intake and mitigates the loss of alcohol preference in mice given continuous HFD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic Variability in Adenosine Deaminase-Like Contributes to Variation in Alcohol Preference in Mice.

A substantial part of the risk for alcohol use disorder is determined by genetic factors. We previously used chromosome substitution (CSS) mice, to identify a quantitative trait loci (QTL) for alcohol preference on mouse chromosome 2. The aim of this study was to identify candidate genes within this QTL that confer the risk for alcohol preference. In order to delineate the neurobiological underpinnings of alcohol consumption, we expanded on the QTL approach to identify candidate genes for high alcohol preference in mice. We narrowed down a QTL for alcohol preference on mouse chromosome 2, that we previously identified using CSS mice, to 4 candidate genes in silico. Expression levels of these candidate genes in prefrontal cortex, amygdala, and nucleus accumbens-brain regions implicated in reward and addiction-were subsequently compared for the CSS-2 and the C57BL/6J host strain. We observed increased expression of adenosine deaminase-like (Adal) in all 3 regions in CSS-2 mice. Moreover, we found that the adenosine deaminase inhibitor EHNA reduced the difference in alcohol preference between CSS-2 and C57BL/6J mice. This study identifies Adal as a genetically protective factor against alcohol consumption in mice, in which elevated Adal levels contribute to low alcohol preference.

FGF21 Regulates Sweet and Alcohol Preference

Fibroblast growth factor 21 (FGF21) is a hormone induced by various metabolic stresses, including ketogenic and high-carbohydrate diets, that regulates energy homeostasis. In humans, SNPs in and around the FGF21 gene have been associated with macronutrient preference, including carbohydrate, fat, and protein intake. Here we show that FGF21 administration markedly reduces sweet and alcohol preference in mice and sweet preference in cynomolgus monkeys. In mice, these effects require the FGF21 co-receptor β-Klotho in the central nervous system and correlate with reductions in dopamine concentrations in the nucleus accumbens. Since analogs of FGF21 are currently undergoing clinical evaluation for the treatment of obesity and type 2 diabetes, our findings raise the possibility that FGF21 administration could affect nutrient preference and other reward behaviors in humans.

Relaxin-3 receptor (RXFP3) signalling mediates stress-related alcohol preference in mice.

Stressful life events are causally linked with alcohol use disorders (AUDs), providing support for a hypothesis that alcohol consumption is aimed at stress reduction. We have previously shown that expression of relaxin-3 mRNA in rat brain correlates with alcohol intake and that central antagonism of relaxin-3 receptors (RXFP3) prevents stress-induced reinstatement of alcohol-seeking. Therefore the objectives of these studies were to investigate the impact of Rxfp3 gene deletion in C57BL/6J mice on baseline and stress-related alcohol consumption. Male wild-type (WT) and Rxfp3 knockout (KO) (C57/B6JRXFP3TM1/DGen) littermate mice were tested for baseline saccharin and alcohol consumption and preference over water in a continuous access two-bottle free-choice paradigm. Another cohort of mice was subjected to repeated restraint followed by swim stress to examine stress-related alcohol preference. Hepatic alcohol and aldehyde dehydrogenase activity was assessed in mice following chronic alcohol intake and in naive controls. WT and Rxfp3 KO mice had similar baseline saccharin and alcohol preference, and hepatic alcohol processing. However, Rxfp3 KO mice displayed a stress-induced reduction in alcohol preference that was not observed in WT littermates. Notably, this phenotype, once established, persisted for at least six weeks after cessation of stress exposure. These findings suggest that in mice, relaxin-3/RXFP3 signalling is involved in maintaining high alcohol preference during and after stress, but does not appear to strongly regulate the primary reinforcing effects of alcohol.

Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors

Serotonin 2C receptors (5-HT(2C)R) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT(2C)R mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT(2C)R mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT(2C)R, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT(2C)R mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT(2C)R mRNA editing underlie alcohol preference in mice.

Genetic Deficiency in Neprilysin or Its Pharmacological Inhibition Initiate Excessive Stress-Induced Alcohol Consumption in Mice

Both acquired and inherited genetic factors contribute to excessive alcohol consumption and the corresponding development of addiction. Here we show that the genetic deficiency in neprilysin [NEP] did not change the kinetics of alcohol degradation but led to an increase in alcohol intake in mice in a 2-bottle-free-choice paradigm after one single stress stimulus (intruder). A repetition of such stress led to an irreversible elevated alcohol consumption. This phenomenon could be also observed in wild-type mice receiving an orally active NEP inhibitor. We therefore elucidated the stress behavior in NEP-deficient mice. In an Elevated Plus Maze, NEP knockouts crossed more often the area between the arms, implicating a significant stronger stress response. Furthermore, such animals showed a decreased locomotor activity under intense light in a locomotor activity test, identifying such mice to be more responsive in aversive situations than their wild-type controls. Since the reduction in NEP activity itself does not lead to significant signs of an altered alcohol preference in mice but requires an environmental stimulus, our findings build a bridge between stress components and genetic factors in the development of alcoholism. Therefore, targeting NEP activity might be a very attractive approach for the treatment of alcohol abuse in a society with increasing social and financial stress.

Effects of Cross‐Fostering on Alcohol Preference and Correlated Responses to Selection in High‐ and Low‐Alcohol‐Preferring Mice

Selectively bred rodent lines are valuable tools for investigating gene×environment interactions related to risk for alcoholism in humans. Early maternal environment is one particular factor known for critically influencing neural, hormonal, and behavioral outcomes in adulthood. Cross-fostering is a procedure that may be used to explore the role of genotype-dependent maternal influences on phenotypic variability in adulthood. The purpose of these experiments was to examine the effects of cross-fostering on free-choice alcohol drinking and correlated responses to selection for alcohol preference in mice selectively bred for high (HAP2) and low (LAP2) alcohol preference. Mice were assigned to one of the following treatments: SHAM (pups that were fostered to their original biological mother), IN (pups that were fostered to a different mother of the same line), and CROSS (pups that were fostered to a mother of a different line). Mice were tested in adulthood for (i) free 24-hour access to alcohol for a period of 28days; (ii) the expression of the acoustic startle response and fear-potentiated startle (FPS); and (iii) handling-induced convulsions (HICs) during acute alcohol withdrawal. Overall, the expression of the alcohol preference selection phenotype was robust in all groups (HAP2>LAP2). Cross-fostering produced a moderate but significant reduction in g/kg alcohol drinking and preference scores in HAP2 mice (CROSS<SHAM) but had no effect in LAP2 mice. Cross-fostering did not affect the expression of correlated responses to selection: acoustic startle response (HAP2>LAP2), FPS (HAP2>LAP2), HICs (LAP2>HAP2). It appears that maternal environment can modify the expression of the high-alcohol-preference phenotype in HAP2 selectively bred mice. These results suggest a gene×environment interaction with respect to the expression of the high-alcohol-preference selection phenotype but not correlated responses to selection.

Tryptophan in alcoholism treatment I: kynurenine metabolites inhibit the rat liver mitochondrial low Km aldehyde dehydrogenase activity, elevate blood acetaldehyde concentration and induce aversion to alcohol.

Aims: The aims were to provide proofs of mechanism and principle by establishing the ability of kynurenine metabolites to inhibit the liver mitochondrial low Km aldehyde dehydrogenase (ALDH) activity after administration and in vivo, and to induce aversion to alcohol. Methods: Kynurenic acid (KA), 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) were administered to normal male Wistar rats and ALDH activity was determined both in vitro in liver homogenates and in vivo (by measuring blood acetaldehyde following ethanol administration). Alcohol consumption was studied in an aversion model in rats and in alcohol-preferring C57 mice. Results: ALDH activity was significantly inhibited by all three metabolites by doses as small as 1 mg/kg body wt. Blood acetaldehyde accumulation after ethanol administration was strongly elevated by KA and 3-HK and to a lesser extent by 3-HAA. All three metabolites induced aversion to alcohol in rats and decreased alcohol preference in mice. Conclusions: The above kynurenine metabolites of tryptophan induce aversion to alcohol by inhibiting ALDH activity. An intellectual property covering the use of 3-HK and 3-HAA and derivatives thereof in the treatment of alcoholism by aversion awaits further development.

Delineation of the Role of Nicotinic Acetylcholine Receptor Genes in Alcohol Preference in Mice

The genetic factors that increase risk for alcohol and nicotine addiction have been elusive, although the frequent co-abuse of these drugs suggests they may act on a common biological pathway. A site of action for both nicotine and alcohol effects in the brain are neuronal nicotinic acetylcholine receptors (nAChR). This report explores the association between six nAChR subunit genes (Chrna3, Chrna4, Chrnb4, Chrnb2, Chrna5, and Chrna7) with alcohol preference (AP) using co-segregation of AP with nAChR subunit genotypes in a F(2) population produced from reciprocal crosses of alcohol-preferring C57BL/6J (B6) and alcohol-avoiding DBA/2J (D2) strains of mice. Polymorphisms located within the Chrna5-Chrna3-Chrnb4 cluster on mouse chromosome 9 were found to co-segregate with AP, with high-drinking F(2) mice carrying B6 alleles and low-drinking F(2) mice carrying D2 alleles. Further, the Chrnb4 and Chrna5 genes showed expression differences between B6 and D2 mice, which is compatible with their involvement in AP in mice and, potentially, alcohol abuse in humans.

Ethanol‐Responsive Genes (Crtam, Zbtb16, and Mobp) Located in the Alcohol‐QTL Region of Chromosome 9 Are Associated With Alcohol Preference in Mice

Previously, our group identified cytotoxic and regulatory T-cell molecule (Crtam), zinc finger and BTB domain containing 16 (Zbtb16), and myelin-associated oligodendrocytic basic protein (Mobp) as ethanol-responsive genes in the mouse brain by gene expression profiling. In this study, we used a genetic co-segregation analysis to assess the association of Crtam, Zbtb16, and Mobp with the alcohol preference (AP) phenotype in the alcohol-preferring C57BL/6J (B6) and alcohol avoiding DBA/2J (D2) strains of mice. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to confirm previous microarray analysis results that Crtam, Zbtb16, and Mobp brain mRNA levels in the B6 and D2 strains are altered by ethanol treatment. The association of the 3 genes with AP was assessed in a F(2) population (n = 427) derived from the reciprocal crosses involving the B6 and D2 strains. Each F(2) individual was assessed for their AP using the 2 bottle choice test and genotyped for Crtam, Zbtb16, and Mobp single nucleotide polymorphisms (SNPs) that differ between B6 and D2 mice. Semi-quantitative RT-PCR analysis confirmed that Crtam, Zbtb16, and Mobp are ethanol-responsive genes. The SNP analyses show that alleles of the 3 genes co-segregate with the AP phenotype in F(2) mice, where individuals homozygous for the B6 allele have higher AP than those homozygous for the D2 allele. Also, the Crtam-Zbtb16 loci that are tightly linked and the Mobp locus act in an additive fashion in determining the relative AP phenotype. Our results are consistent with the hypothesis that Crtam, Zbtb16, and Mobp may be involved in AP in mice. The nature of this association remains to be established and may reflect a direct effect of these genes or an indirect effect caused by linked genes on mouse chromosome 9.

Studies on Syntaxin 12 and Alcohol Preference Involving C57BL/6J and DBA/2J Strains of Mice

C57BL/6J and DBA/2J inbred mouse strains have been extensively studied for the genetic dissection of alcohol-related phenotypes. We have previously found Syntaxin 12 to be associated with alcohol preference in C57BL/6J and DBA/2J due to its strain-specific and ethanol responsive expression in the male brain. In the current study, we combined genetic and expression analyses to assess the segregation of Syntaxin 12 c.*1370G>A polymorphism with its strain-specific expression and alcohol preference in an F (2) population (N = 427) derived from C57BL/6J and DBA/2J strains. Syntaxin 12 c.*1370G>A polymorphism was found to segregate with alcohol preference in the B6D2F2 population and a correlation was identified between Syntaxin 12 expression and alcohol preference in the selected B6D2F2 males (r = -0.473, r (2) = 0.22). We followed up our analysis in the BXD RI lines using resources from WebQTL and the Mouse Phenome Database. Our study detected significant associations of Syntaxin 12 molecular variants with its level of expression and alcohol preference in B6D2F2 males. Overall, our findings support a role for Syntaxin 12 as a potential contributor to alcohol preference in mice.

The genomic determinants of alcohol preference in mice

Searches for the identity of genes that influence the levels of alcohol consumption by humans and other animals have often been driven by presupposition of the importance of particular gene products in determining positively or negatively reinforcing effects of ethanol. We have taken an unbiased approach and performed a meta-analysis across three types of mouse populations to correlate brain gene expression with levels of alcohol intake. Our studies, using filtering procedures based on QTL analysis, produced a list of eight candidate genes with highly heritable expression, which could explain a significant amount of the variance in alcohol preference in mice. Using the Allen Brain Atlas for gene expression, we noted that the candidate genes' expression was localized to the olfactory and limbic areas as well as to the orbitofrontal cortex. Informatics techniques and pathway analysis illustrated the role of the candidate genes in neuronal migration, differentiation, and synaptic remodeling. The importance of olfactory cues, learning and memory formation (Pavlovian conditioning), and cortical executive function, for regulating alcohol intake by animals (including humans), is discussed.

Involvement of cannabinoid CB2 receptor in alcohol preference in mice and alcoholism in humans

We tested if cannabinoid type 2 receptor (CB2) in the central nervous system plays a role in alcohol abuse/dependence in animal model and then examined an association between the CB2 gene polymorphism and alcoholism in human. Mice experiencing more alcohol preference by drinking showed reduced Cb2 gene expression, whereas mice with little preference showed no changes of it in ventral midbrain. Alcohol preference in conjunction with chronic mild stress were enhanced in mice treated with CB2 agonist JWH015 when subjected to chronic stress, whereas antagonist AM630 prevented development of alcohol preference. There is an association between the Q63R polymorphism of the CB2 gene and alcoholism in a Japanese population (P=0.007; odds ratio 1.25, 95% CI, (1.06-1.47)). CB2 under such environment is associated with the physiologic effects of alcohol and CB2 antagonists may have potential as therapies for alcoholism.

Cyclosporine-A discourages ethanol intake in C57bl/6j mice: a preliminary study.

Remarkably high rates of abstinence occur among alcohol-dependent persons after a liver transplant. This was thought to be solely due to selection or other nonbiological factors. Observing sustained abstinence for many months to years after a liver transplant, however, we asked whether immunosuppressants might exert a biological effect that supported abstinence from ethanol. We hypothesized that cyclosporine-A-treated mice would drink less ethanol given a choice between water and 10% ethyl alcohol. After a 3-day ethanol conditioning period, C57bl/6j mice (n = 20) medicated with cyclosporine-A (50 mg/kg) and a control group of unmedicated mice (n = 20) injected with saline were given free access to either water or the ethanol solution. The medicated mice drank significantly less ethanol throughout the 11-day choice protocol. This was significant whether measured by mean alcohol consumption per day (p = .003) or by alcohol preference as a percent of total liquid intake per day (p = .03). The two groups did not differ significantly in total daily fluid consumption. These data suggest that cyclosporine-A lowers alcohol preference in mice. Mechanism of action, similar effects by other centrally acting immunosuppressants and translation to humans all remain to be studied. If fruitful, elucidating the actions of cyclosporine and other immunosuppressants that activate central nervous system receptors may illuminate the pathophysiology of alcohol use and addiction.

Approaches to Understanding the Neurobiological Regulation of Ethanol Self‐Administration: A Young Investigators Forum

The article represents the proceedings of a symposium at the 2000 RSA Meeting in Denver, Colorado. The co-chairs were Cristine L. Czachowski and Craig J. Slawecki. The presentations were (1) Behavioral assessment of ethanol seeking and self-administration, by Cristine L. Czachowski; (2) Electrophysiological measures of ethanol preference and reinforcing efficacy, by Craig J. Slawecki; (3) Genetic differences in locomotor sensitization and reward phenotypes associated with bidirectional selection for alcohol preference in mice, by Nicholas J. Grahame; (4) Transgenic and knockout mouse models: Powerful tools for investigating the neurobiology of alcoholism, by Todd E. Thiele; and (5) Neurochemical characteristics that may predict ethanol preference in selected P and NP rats: A quantitative microdialysis study, by Simon N. Katner.

Approaches to Understanding the Neurobiological Regulation of Ethanol Self-Administration: A Young Investigators Forum

The article represents the proceedings of a symposium at the 2000 RSA Meeting in Denver, Colorado. The co-chairs were Cristine L. Czachowski and Craig J. Slawecki. The presentations were (1) Behavioral assessment of ethanol seeking and self-administration, by Cristine L. Czachowski; (2) Electrophysiological measures of ethanol preference and reinforcing efficacy, by Craig J. Slawecki; (3) Genetic differences in locomotor sensitization and reward phenotypes associated with bidirectional selection for alcohol preference in mice, by Nicholas J. Grahame; (4) Transgenic and knockout mouse models: Powerful tools for investigating the neurobiology of alcoholism, by Todd E. Thiele; and (5) Neurochemical characteristics that may predict ethanol preference in selected P and NP rats: A quantitative microdialysis study, by Simon N. Katner.

Epistatic quantitative trait loci for alcohol preference in mice.

Alcohol consumption is a complex trait, responding to the influence of various genes and environmental influences acting in a quantitative fashion. Various studies in alcohol consumption processes have identified quantitative trait locus (QTL) regions across the mouse genome that appear to contribute to this phenotype. The purpose of this study was to examine the influence of interactions between alleles at different loci, a phenomenon known as epistasis, on previously identified QTLs for alcohol consumption in mice. A multiple regression model was developed and applied to test for the significance of the interaction between two QTLs and to quantify this interaction. Our results indicate the presence of epistasis between loci on mouse chromosomes 2 and 3 accounting for 7-8% of the variation in alcohol preference, respectively.

Selective breeding for high and low alcohol preference in mice.

High and low alcohol preference (HAP and LAP, respectively) mice were created by 10 generations of bidirectional selection for differences in two-bottle choice alcohol consumption. The progenitors used for selection were HS/lbg mice, which are a genetically defined, outbred stock. During selection, mice had 24-h, daily access to 10% alcohol (v/v) and water ad libitum for 30 days and were selected based on the alcohol (g/kg) consumed per day over the entire period. Food was available ad libitum. At S10, line means for alcohol consumption differed greatly, with consumption of over 12 g/kg per day in the HAP mice and less than 2 g/kg per day in the LAP mice. Realized heritability for bidirectional selection was approximately 0.2. Female mice consumed more alcohol than male mice. There were no differences between lines in alcohol elimination rate, nor were there line differences in intake of salt or quinine solutions. However, consumption of saccharin solutions was greater in HAP mice than LAP mice, consistent with previous findings of a genetic correlation between sweet preference and alcohol drinking. Because the mouse genome is relatively well characterized, these selected lines should prove a useful tool for assessment of the genetic basis of, and phenotypes that correlate with, alcohol drinking.

Genomic screen for QTLs underlying alcohol consumption in the P and NP rat lines

Selective breeding for voluntary alcohol consumption was utilized to establish the alcohol-preferring (P) and alcohol-nonpreferring (NP) rat lines. Inbreeding was initiated after 30 generations of selection and, after 19 generations of inbreeding, 384 F2 intercross progeny were created to identify quantitative trait loci (QTLs) influencing alcohol consumption. We had reported previously a QTL on Chromosome (Chr) 4; additional markers genotyped on Chr 4 have increased the maximum lod score from 8.6 to 9.2. This QTL acts in an additive fashion and continues to account for approximately 11% of the phenotypic variability. The 95% confidence interval is 12.5 cM and includes the candidate gene, neuropeptide Y. Subsequent to the identification of the QTL on Chr 4, a genome scan was completed to identify additional QTLs influencing alcohol consumption. A lod score of 2.5 was obtained on Chr 3, syntenic to a region previously reported for alcohol preference in mice. Analysis of Chr 8 produced a lod score of 2.2 near the dopamine D2 and serotonin 1b receptors, which have been previously reported as candidate genes for alcohol preference. Evidence for linkage to alcohol consumption was not found on any other chromosome. It therefore appears likely that, in addition to the QTL on Chr 4, multiple loci of small to moderate effect, such as those on Chrs 3 and 8, underlie the difference in alcohol consumption in the P/NP lines.