Nonpreferring Research Articles

Evaluating habit formation across pairs of female and male selectively bred alcohol-preferring and non-preferring rats

Some individuals with alcohol use disorder (AUD) continue to drink because they have developed a habit where they do not consider the consequences of their actions. Genetically selected lines of alcohol-preferring and non-preferring rats allow for exploration of how specific endophenotypes, such as tendency to form habits, may be risk factors that interact with a genetic predisposition of AUD. While high alcohol drinking (HAD) and alcohol-preferring (P) rats were selectively bred to consume high amounts of freely available ethanol, they exhibit differences in alcohol-seeking behaviors as well as impulsive behaviors, and may represent different behavioral models of AUD. The goal of the current study was to compare the tendency to develop habitual behaviors across female and male HAD1, HAD2, and P rats and their respective alcohol non-preferring counterparts. Alcohol-naïve rats were trained on a variable interval schedule using a non-ethanol reinforcer and were then tested in two extinction sessions, one prior to a reinforcer devaluation (conditioned taste aversion) procedure and one after. There were no differences in total lever presses between P and alcohol non-preferring (NP) rats, but there were differences between HAD and low-alcohol drinking (LAD) rats. All six strains decreased lever pressing after reinforcer devaluation. However, P and NP females did not increase latency to first lever press after devaluation, suggesting some inclination toward habitual behavior that was not apparent in either the HAD or LAD lines. Selective breeding for alcohol preference does not seem to influence the tendency to form habits, whereas background strain and sex may have an influence on this behavior.

Epigenetic changes on rat chromosome 4 contribute to disparate alcohol drinking behavior in alcohol-preferring and -nonpreferring rats.

Paternal alcohol abuse is a well-recognized risk factor for the development of an alcohol use disorder (AUD). In addition to genetic and environmental risk factors, heritable epigenetic factors also have been proposed to play a key role in the development of AUD. However, it is not clear whether epigenetic factors contribute to the genetic inheritance in families affected by AUD. We used reciprocal crosses of the alcohol-preferring (P) and -nonpreferring (NP) rat lines to test whether epigenetic factors also impacted alcohol drinking in up to two generations of offspring. F1 offspring derived by reciprocal breeding of P and NP rats were tested for differences in alcohol consumption using a free-choice protocol of 10% ethanol, 20% ethanol, and water that were available concurrently. In a separate experiment, an F2 population was tested for alcohol consumption not only due to genetic differences. These rats were generated from inbred P (iP) and iNP rat lines that were reciprocally bred to produce genetically identical F1 offspring that remained alcohol-naïve. Intercrosses of the F1 generation animals produced the F2 generation. Alcohol consumption was then assessed in the F2 generation using a standard two-bottle choice protocol, and was analyzed using genome-wide linkage analysis. Alcohol consumption measures were also analyzed for sex differences. Average alcohol consumption was higher in the F1 offspring of P vs. NP sires and in the F2 offspring of F0 iP vs. iNP grandsires. Linkage analyses showed the maximum LOD scores for alcohol consumption in both male and female offspring were on chromosome 4 (Chr 4). The LOD score for both sexes considered together was higher when the grandsire was iP vs. iNP (5.0 vs. 3.35, respectively). Furthermore, the F2 population displayed enhanced alcohol consumption when the P alleles from the F0 sire were present. These results demonstrate that epigenetic and/or non-genetic factors mapping to rat chromosome 4 contribute to a transgenerational paternal effect on alcohol consumption in the P and NP rat model of AUD.

Estrogen-Dependent Upregulation of Adcyap1r1 Expression in Nucleus Accumbens Is Associated With Genetic Predisposition of Sex-Specific QTL for Alcohol Consumption on Rat Chromosome 4.

Humans show sex differences related to alcohol use disorders (AUD). Animal model research has the potential to provide important insight into how sex differences affect alcohol consumption, particularly because female animals frequently drink more than males. In previous work, inbred strains of the selectively bred alcohol-preferring (P) and non-preferring (NP) rat lines revealed a highly significant quantitative trait locus (QTL) on rat chromosome 4, with a logarithm of the odds score of 9.2 for alcohol consumption. Recently, interval-specific congenic strains (ISCS) were developed by backcrossing the congenic P.NP line to inbred P (iP) rats to further refine the chromosome 4 QTL region. Two ISCS sub-strains, ISCS-A and ISCS-B, were obtained with a narrowed QTL, where the smallest region of overlap consisted of 8.9 Mb in ISCS-B. Interestingly, we found that females from both ISCS lines consumed significantly less alcohol than female iP controls (p < 0.05), while no differences in alcohol consumption were observed between male ISCS and iP controls. RNA-sequencing was performed on the nucleus accumbens of alcohol-naïve female ISCS-B and iP rats, which revealed differentially expressed genes (DEG) with greater than 2-fold change and that were functionally relevant to behavior. These DEGs included down-regulation of Oxt, Asb4, Gabre, Gabrq, Chat, Slc5a7, Slc18a8, Slc10a4, and Ngfr, and up-regulation of Ttr, Msln, Mpzl2, Wnt6, Slc17a7, Aldh1a2, and Gstm2. Pathway analysis identified significant alterations in gene networks controlling nervous system development and function, as well as cell signaling, GABA and serotonin receptor signaling and G-protein coupled receptor signaling. In addition, β-estradiol was identified as the most significant upstream regulator. The expression levels of estrogen-responsive genes that mapped to the QTL interval and have been previously associated with alcohol consumption were measured using RT-qPCR. We found that expression of the Adcyap1r1 gene, encoding the pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor, was upregulated in female ISCS-B compared to female iP controls, while no differences were exhibited in males. In addition, sequence variants in the Adcyap1r1 promoter region showed a differential response to estrogen stimulation in vitro. These findings demonstrate that rat chromosome 4 QTL contains genetic variants that respond to estrogen and are associated with female alcohol consumption.

Delay discounting for sucrose in alcohol-preferring and nonpreferring rats using a sipper tube within-sessions task.

Delay discounting (DD) is a measure of impulsivity that quantifies preference for a small reward delivered immediately over a large reward delivered after a delay. It has been hypothesized that impulsivity is an endophenotype associated with increased risk for development of alcohol use disorders (AUDs); however, a causal role of impulsivity is difficult to determine with human studies. We tested this hypothesis by assessing the degree of DD present in alcohol-naïve rats selectively bred for either high- or low-alcohol preference. A novel adaptation of a within-sessions DD procedure was used to compare impulsivity differences between male alcohol-preferring (P) and nonpreferring (NP) rat lines (n=6 per line) using a 5% sucrose reward. Animals chose between 2 options: 2-second sipper tube access delivered immediately (small reward) or 8-second access after a variable delay (large reward). Each 50-minute session consisted of 5 blocks of ten 60-second trials. Within each session, the delay to the large reward increased in each block of trials. Delays were gradually increased over 3 sets to attain a final delay set of 3, 8, 15, 18, and 25seconds. Prior to starting delays, there were no significant differences between lines in sucrose consumption or percent choice for the large reward, and both lines exhibited a clear preference for the large reward. After delays were initiated, choice for the large reward decreased as the delay to its presentation increased. Although discounting of the large, delayed reward was observed for both lines, the degree of discounting, or "impulsivity," was greater for P rats compared with NP rats. P rats are more impulsive for sucrose rewards before exposure to alcohol compared with NP rats. Thus, individuals genetically predisposed toward developing AUDs may be more likely to engage in impulsive decision making prior to alcohol exposure.

Effects of histone deacetylase inhibitors on amygdaloid histone acetylation and neuropeptide Y expression: a role in anxiety-like and alcohol-drinking behaviours

Recent studies have demonstrated the involvement of epigenetic mechanisms in psychiatric disorders, including alcoholism. Here, we investigated the effects of histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) on amygdaloid HDAC-induced histone deacetylation and neuropeptide Y (NPY) expression and on anxiety-like and alcohol-drinking behaviours in alcohol-preferring (P) and -non-preferring (NP) rats. It was found that P rats displayed higher anxiety-like and alcohol-drinking behaviours, higher amygdaloid nuclear, but not cytosolic, HDAC activity, which was associated with increased HDAC2 protein levels and deficits in histone acetylation and NPY expression in the central (CeA) and medial nucleus of amygdala (MeA), as compared to NP rats. TSA treatment attenuated the anxiety-like and alcohol-drinking behaviours, with concomitant reductions in amygdaloid nuclear, but not cytosolic HDAC activity, and HDAC2, but not HDAC4, protein levels in the CeA and MeA of P rats, without effect in NP rats. TSA treatment also increased global histone acetylation (H3-K9 and H4-K8) and NPY expression in the CeA and MeA of P, but not in NP rats. Histone H3 acetylation within the NPY promoter was also innately lower in the amygdala of P rats compared with NP rats; which was normalized by TSA treatment. Voluntary ethanol intake in P, but not NP rats, produced anxiolytic effects and decreased the HDAC2 levels and increased histone acetylation in the CeA and MeA. These results suggest that higher HDAC2 expression-related deficits in histone acetylation may be involved in lower NPY expression in the amygdala of P rats, and operative in controlling anxiety-like and alcohol-drinking behaviours.

Changes in gene expression within the ventral tegmental area following repeated excessive binge-like alcohol drinking by alcohol-preferring (P) rats

The objective of this study was to detect changes in gene expression in the ventral tegmental area (VTA) following repeated excessive binge-like (‘loss-of-control’) alcohol drinking by alcohol-preferring (P) rats. Adult female P rats (n = 7) were given concurrent access to 10, 20, and 30% EtOH for 4 1-h sessions daily for 10 weeks followed by 2 cycles of 2 weeks of abstinence and 2 weeks of EtOH access. Rats were sacrificed by decapitation 3 h after the 4th daily EtOH-access session at the end of the second 2-week relapse period. A water-control group of female P rats (n = 8) was also sacrificed. RNA was prepared from micro-punch samples of the VTA from individual rats; analyses were conducted with Affymetrix Rat 230.2 GeneChips. Ethanol intakes were 1.2–1.7 g/kg per session, resulting in blood levels >200 mg% at the end of the 4th session. There were 211 unique named genes that significantly differed (FDR = 0.1) between the water and EtOH groups. Bioinformatics analyses indicated alterations in a) transcription factors that reduced excitation-coupled transcription and promoted excitotoxic neuronal damage involving clusters of genes associated with Nfkbia, Fos, and Srebf1, b) genes that reduced cholesterol and fatty acid synthesis, and increased protein degradation, and c) genes involved in cell-to-cell interactions and regulation of the actin cytoskeleton. Among the named genes, there were 62 genes that showed differences between alcohol-naïve P and non-preferring (NP) rats, with 43 of the genes changing toward NP-like expression levels following excessive binge-like drinking in the P rats. These genes are involved in a pro-inflammatory response, and enhanced response to glucocorticoids and steroid hormones. Overall, the results of this study indicate that the repeated excessive binge-like alcohol drinking can change the expression of genes that may alter neuronal function in several ways, some of which may be deleterious.

Aberrant Histone Deacetylase2–Mediated Histone Modifications and Synaptic Plasticity in the Amygdala Predisposes to Anxiety and Alcoholism

Epigenetic mechanisms have been implicated in psychiatric disorders, including alcohol dependence. However, the epigenetic basis and role of specific histone deacetylase (HDAC) isoforms in the genetic predisposition to anxiety and alcoholism is unknown. We measured amygdaloid HDAC activity, levels of HDAC isoforms, and histone H3 acetylation in selectively bred alcohol-preferring (P) and -nonpreferring (NP) rats. We employed HDAC2 small interfering RNA infusion into the central nucleus of amygdala (CeA) of P rats to determine the causal role of HDAC2 in anxiety-like and alcohol-drinking behaviors. Chromatin immunoprecipitation analysis was performed to examine the histone acetylation status of brain-derived neurotrophic factor (Bdnf) and activity-regulated cytoskeleton associated protein (Arc) genes. Golgi-Cox staining was performed to measure dendritic spine density. We found that P rats innately display higher nuclear HDAC activity and HDAC2 but not HDAC 1, 3, 4, 5, and 6 protein levels and lower acetylation of H3-K9 but not H3-K14, in the CeA and medial nucleus of amygdala compared with NP rats. Acute ethanol exposure decreased amygdaloid HDAC activity and HDAC2 protein levels, increased global and gene (Bdnf and Arc)-specific histone acetylation, and attenuated anxiety-like behaviors in P rats but had no effects in NP rats. The HDAC2 knockdown in the CeA attenuated anxiety-like behaviors and voluntary alcohol but not sucrose consumption in P rats and increased histone acetylation of Bdnf and Arc with a resultant increase in protein levels that correlated with increased dendritic spine density. These novel data demonstrate the role of HDAC2-mediated epigenetic mechanisms in anxiety and alcoholism.

Ethanol affects acylated and total ghrelin levels in peripheral blood of alcohol-dependent rats

There is a hypothesis that ghrelin could take part in the central effects of alcohol as well as function as a peripheral indicator of the changes which occur during long-term alcohol consumption. The aim of this study was to determine a correlation between alcohol concentration and acylated and total form of ghrelin after a single administration of alcohol (intraperitoneal, i.p.) (experiment 1) and prolonged ethanol consumption (experiment 2). The study was performed using Wistar alcohol preferring (PR) and non-preferring (NP) rats and rats from inbred line (Warsaw High Preferring, WHP; Warsaw Low Preferring, WLP). It was found that ghrelin in ethanol-naive WHP animals showed a significantly lower level when compared with the ethanol-naive WLP or Wistar rats. After acute ethanol administration in doses of 1.0; 2.0 and 4.0 g/kg, i.p., the simple (WHP) or inverse (WLP and Wistar) relationship between alcohol concentration and both form of ghrelin levels in plasma were found. Chronic alcohol intake in all groups of rats led to decrease of acylated ghrelin concentration. PR and WHP rats, after chronic alcohol drinking, had lower levels of both form of ghrelin in comparison with NP and WLP rats, respectively, and the observed differences in ghrelin levels were in inverse relationship with their alcohol intake. In conclusion, it is suggested that there is a strong relationship between alcohol administration or intake, ethanol concentration in blood and both active and total ghrelin level in the experimental animals, and that ghrelin plasma concentration can be a marker of alcohol drinking predisposition.

Quantitative trait locus for body weight identified on rat chromosome 4 in inbred alcohol-preferring and -nonpreferring rats: Potential implications for neuropeptide Y and corticotrophin releasing hormone 2

The alcohol-preferring (P) and -nonpreferring (NP) rat lines were developed using bidirectional selective breeding for alcohol consumption (g/kg/day) and alcohol preference (water:ethanol ratio). During a preliminary study, we detected a difference in body weight between inbred P (iP) and inbred NP (iNP) rats that appeared to be associated with the transfer of the Chromosome 4 quantitative trait locus (QTL) seen in the P.NP and NP.P congenic strains. After the initial confirmation that iP rats displayed lower body weight when compared to iNP rats (data not shown), body weight and growth rates of each chromosome 4 reciprocal congenic rat strain (P.NP and NP.P) were measured, and their body weight was consistent with their respective donor strain phenotype, confirming that a quantitative trait locus for body weight mapped to the chromosome 4 interval. Utilizing the newly developed interval-specific congenic strains (ISCS-A and ISCS-B), the QTL interval was further narrowed identifying the following candidate genes of interest: neuropeptide Y (Npy), juxtaposed with another zinc finger gene 1 (Jazf1), corticotrophin releasing factor receptor 2 (Crfr2) and LanC lantibiotic synthetase component C-like 2 (Lancl2). These findings indicate that a biologically active variant(s) regulates body weight on rat chromosome 4 in iP and iNP rats. This QTL for body weight was successfully captured in the P.NP and NP.P congenic strains, and interval-specific congenic strains (ISCSs) were subsequently employed to fine-map the QTL interval identifying the following candidate genes of interest: Npy, Jazf1, Crfr2 and Lancl2. Both Npy and Crfr2 have been previously identified as candidate genes of interest underlying the chromosome 4 QTL for alcohol consumption in iP and iNP rats.

GABAergic Gene Expression in Postmortem Hippocampus from Alcoholics and Cocaine Addicts; Corresponding Findings in Alcohol-Naïve P and NP Rats

BackgroundBy performing identical studies in humans and rats, we attempted to distinguish vulnerability factors for addiction from neurobiological effects of chronic drug exposure. We focused on the GABAergic system within the hippocampus, a brain region that is a constituent of the memory/conditioning neuronal circuitry of addiction that is considered to be important in drug reinforcement behaviors in animals and craving and relapse in humans.MethodologyUsing RNA-Seq we quantified mRNA transcripts in postmortem total hippocampus from alcoholics, cocaine addicts and controls and also from alcohol-naïve, alcohol preferring (P) and non-preferring (NP) rats selectively bred for extremes of alcohol-seeking behavior that also show a general addictive tendency. A pathway-targeted analysis of 25 GABAergic genes encoding proteins implicated in GABA synthesis, metabolism, synaptic transmission and re-uptake was undertaken.Principal FindingsDirectionally consistent and biologically plausible overlapping and specific changes were detected: 14/25 of the human genes and 12/25 of the rat genes showed nominally significant differences in gene expression (global p values: 9×10−14, 7×10−11 respectively). Principal FDR-corrected findings were that GABBR1 was down-regulated in alcoholics, cocaine addicts and P rats with congruent findings in NSF, implicated in GABAB signaling efficacy, potentially resulting in increased synaptic GABA. GABRG2, encoding the gamma2 subunit required for postsynaptic clustering of GABAA receptors together with GPHN, encoding the associated scaffolding protein gephryin, were both down-regulated in alcoholics and cocaine addicts but were both up-regulated in P rats. There were also expression changes specific to cocaine addicts (GAD1, GAD2), alcoholics (GABRA2) and P rats (ABAT, GABRG3).Conclusions/SignificanceOur study confirms the involvement of the GABAergic system in alcoholism but also reveals a hippocampal GABA input in cocaine addiction. Congruent findings in human addicts and P rats provide clues to predisposing factors for alcohol and drug addiction. Finally, the results of this study have therapeutic implications.

S09 * ALCOHOLISM: TRANSCRIPTIONAL/EPIGENETIC ADAPTATIONS IN ANIMAL AND HUMAN BRAIN * S09.1 * EPIGENETIC BASIS FOR ANXIETY AND ALCOHOLISM

S09 * ALCOHOLISM: TRANSCRIPTIONAL/EPIGENETIC ADAPTATIONS IN ANIMAL AND HUMAN BRAIN * S09.1 * EPIGENETIC BASIS FOR ANXIETY AND ALCOHOLISM

Genetic propensities to increase ethanol intake in response to stress: studies with selectively bred swim test susceptible (SUS), alcohol-preferring (P), and non-preferring (NP) lines of rats

Swim test susceptible (SUS) rats selectively bred for reduced struggling in the forced swim test (FST) following stress show high voluntary ethanol intake like alcohol-preferring (P) rats selectively bred for ethanol preference. It is unknown whether stress enhances drinking in SUS rats or FST behavior in P and non-preferring (NP) rats. The aim of this study was to assess the response to stress in male SUS, Sprague-Dawley (SD), P, and NP rats on 10% ethanol drinking and FST behavior. In experiment 1, SUS and SD rats had limited access to ethanol and water following white noise, rehousing, and forced swim stress. In experiment 2, P and NP rats received footshock, white noise, restraint, or no stress prior to the FST. Rats then had continuous access to ethanol and water, and the effects of weekly exposures to stress were measured. SUS rats drank more ethanol (M = 2.98 g/kg) than SD rats (M = 1.26 g/kg) at baseline. Stress produced sustained increases (~33% of baseline) in ethanol intake in SUS rats. NP rats spent twice as much time immobile as P rats in the FST. Stress did not alter FST behavior in P or NP rats. Only footshock produced an increase (~29%) in ethanol intake in P rats. Selection for stress-induced depressive-like behavior in SUS rats is associated with enhanced stress-induced ethanol drinking. However, the selection for alcohol preference is not associated with stress-induced depressive-like behavior but is associated with footshock stress-induced ethanol drinking. In these experiments, relationships among stress, depressive-like behavior, and alcohol preference were not symmetrical.

Differential rearing conditions and alcohol-preferring rats: Consumption of and operant responding for ethanol.

Exposing rats to differential rearing conditions during early postweaning development has been shown to produce changes in a number of behaviors displayed during adulthood. The purpose of the present studies was to investigate whether rearing alcohol-preferring (P) and nonpreferring (NP) rats in an environmental enrichment condition (EC), a social condition (SC), or an impoverished condition (IC) would differentially affect self-administration of 10% ethanol. In Experiment 1, rats were tested for consumption of 10% ethanol in limited- and free-access tests. For Experiment 2, rats were trained to respond in an operant chamber for ethanol and then provided concurrent access to 10% ethanol and water. Each solution was presented in a separate liquid dipper after meeting the schedule of reinforcement on distinct levers. After concurrent access tests, the water lever/dipper was inactivated and a progressive ratio (PR) schedule was initiated. Three successive solutions (10% ethanol, 15% ethanol, and 10% sucrose) were tested under the PR. For P rats, rearing in an EC reduced ethanol consumption, preference, and motivation to obtain ethanol, relative to P rats reared in an IC. Thus, exposure to a novel environment immediately after weaning acted to decrease the reinforcing properties of ethanol in an animal model for alcoholism.

The role of amygdaloid brain‐derived neurotrophic factor, activity‐regulated cytoskeleton‐associated protein and dendritic spines in anxiety and alcoholism

Innate anxiety appears to be a robust factor in the promotion of alcohol intake, possibly due to the anxiolytic effects of self-medication with alcohol. Brain-derived neurotrophic factor (BDNF) and its downstream target, activity-regulated cytoskeleton-associated (Arc) protein, play a role in the regulation of synaptic function and structure. In order to examine the role of the BDNF-Arc system and associated dendritic spines in the anxiolytic effects of ethanol, we investigated the effects of acute ethanol exposure on anxiety-like behaviors of alcohol-preferring (P) and -nonpreferring (NP) rats. We also examined changes in the expression of BDNF and Arc, and dendritic spine density (DSD), in amygdaloid brain regions of P and NP rats with or without ethanol exposure. It was found that in comparison with NP rats, P rats displayed innate anxiety-like behaviors, and had lower mRNA and protein levels of both BDNF and Arc, and also had lower DSD in the central amygdala (CeA) and medial amygdala (MeA), but not in the basolateral amygdala (BLA). Acute ethanol treatment had an anxiolytic effect in P, but not in NP rats, and was associated with an increase in mRNA and protein levels of BDNF and Arc, and in DSD in the CeA and MeA, but not BLA. These results suggest that innate deficits in BDNF-Arc levels, and DSD, in the CeA and MeA may be involved in the anxiety-like and excessive alcohol-drinking behaviors of P rats, as ethanol increased these amygdaloid synaptic markers and produced anxiolytic effects in P rats, but not NP rats.

Melanocortin activity in the amygdala influences alcohol intake

Melanocortins have been reported to affect alcohol intake through actions in the hypothalamus thought to be mediated by melanocortin MC4 receptors. Since these receptors are expressed in a number of amygdala regions, we have explored their role in the regulation of alcohol intake in both alcohol-preferring (P) and non-preferring (NP) rats. Injections were made at the border of the central amygdala nucleus and the basolateral amygdala. The MC3/MC4R agonist MTII reduced alcohol and food intake but increased water intake while the selective MC4R antagonist HS014 only increased food and water intake. The MC3/MC4R antagonist SHU9119 increased food and water but had little effect on alcohol intake. However, when the SHU9119 stimulation of food intake was prevented by pair-feeding, SHU9119 induced a large and prolonged decline in alcohol intake that was paralleled by an increase in water intake. These effects were only observed in P rats. We conclude that melanocortin activity in the amygdala can alter the selective preference for water and alcohol independent of effects on food intake.

Ontogeny of ethanol intake in alcohol preferring (P) and alcohol nonpreferring (NP) rats

There is a scarcity of research on ethanol affinity in alcohol-preferring (P) rats before weaning and it is unknown if neonate P rats exhibit ethanol intake preferences comparable to those observed in adult P rats. This study examined ethanol intake in P and alcohol-nonpreferring (NP) rats 3 hr after birth (Experiment 1, surrogate nipple test), at postnatal days (PD) 8, 12, and 18 (Experiment 2, consumption from the floor procedure) and at adolescence (Experiment 3, two-bottle choice test at PD32). The high-preference genotype was readily expressed 3 hr after birth. P neonates drank twice as much ethanol as their NP counterparts. This heightened ethanol preference transiently reversed at P8, reemerged as weaning approached (P18) and was fully expressed during adolescence. These results help to clarify the ontogeny of genetic predisposition for ethanol. Genetic predisposition for higher ethanol intake in P than in NP rats seems to be present immediately following birth.

Sleep–wakefulness in alcohol preferring and non-preferring rats following binge alcohol administration

The alcohol-preferring (P) rat is a valid animal model of alcoholism. However, the effect of alcohol on sleep in P or alcohol non-preferring (NP) rats is unknown. Since alcohol consumption has tremendous impact on sleep, the present study compared the effects of binge alcohol administration on sleep–wakefulness in P and NP rats. Using standard surgical procedures, the P and NP rats were bilaterally implanted with sleep recording electrodes. Following post-operative recovery and habituation, pre-ethanol (baseline) sleep–wakefulness was electrographically recorded for 48 h. Subsequently, ethanol was administered beginning with a priming dose of 5 g/Kg followed by two doses of 2 g/Kg every 8 h on the first day and three doses of 3 g/Kg/8 h on the second day. On the following day (post-ethanol), undisturbed sleep–wakefulness was electrographically recorded for 24 h. Our initial results suggest that, during baseline conditions, the time spent in each of the three behavioral states: wakefulness, non-rapid eye movement (NREM) sleep and REM sleep, was comparable between P and NP rats. However, the P rats were more susceptible to changes in sleep–wakefulness following 2 days of binge ethanol treatment. As compared to NP rats, the P rats displayed insomnia like symptoms including a significant reduction in the amount of time spent in NREM sleep coupled with a significant increase in wakefulness on post-ethanol day. Subsequent analysis revealed that binge ethanol induced increased wakefulness and reduced NREM sleep in P rats occurred mainly in the dark period. This is the first study that: (1) demonstrates spontaneous sleep–wake profile in P and NP rats, and (2) compares the effects of binge ethanol treatment on sleep in P and NP rats. Our results suggest that, as compared to NP rats, the P rats were more susceptible to sleep disruptions after binge ethanol treatment. In addition, the P rats exhibited insomnia-like symptoms observed during abstinence from alcohol in human subjects.

Event-related oscillations in the parietal cortex of adult alcohol-preferring (P) and alcohol-nonpreferring rats (NP)

The selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines were developed from Wistar rats to model high and low voluntary alcohol consumption and have been demonstrated to exhibit many of the characteristics of human alcohol dependence. Electrophysiologic studies have shown P rats exhibit more electroencephalographic fast frequency activity and reduced P3 amplitude in the parietal cortex than NP rats, findings that are more common in alcohol-dependent individuals. Event-related oscillations (EROs) have been suggested to be good endophenotypes associated with ethanol dependence in clinical studies. Recently EROs have also been demonstrated to occur in rodents in response to stimuli that are similar to that used in human clinical studies. The objective of the present study was to characterize EROs in adult P and NP rats. A time-frequency representation method was used to determine delta, theta, and alpha/beta ERO energy and the degree of phase variation in the parietal cortex of adult P and NP rats. The present results suggest that the decrease in P3 amplitudes previously shown in P rats were not associated with changes in ERO energy but were significantly associated with decreases in evoked delta and alpha/beta phase locking. These studies demonstrate ERO measures may also be good endophenotypes in animal models of alcoholism.

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.

From QTL to Candidate Gene: A Genetic Approach to Alcoholism Research

A major focus of research in alcohol-related disorders is to identify the genes and pathways that modulate alcohol-seeking behavior. In light of this, animal models have been established to study various aspects of alcohol dependence. The selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines were developed from Wistar rats to model high and low voluntary alcohol consumption, respectively. Using inbred P and NP strains, a strong QTL (LOD-9.2) for alcohol consumption was identified on rat chromosome 4. To search for candidate genes that underlie this chromosomal region, complementary molecular-based strategies were implemented to identify genetic targets that likely contribute to the linkage signal. In an attempt to validate these genetic targets, corroborative studies have been utilized including pharmacological studies, knock-out/transgenic models as well as human association studies. Thus far, three candidate genes, neuropeptide Y (Npy), alpha-synuclein (Snca), and corticotrophin-releasing factor receptor 2 (Crhr2), have been identified that may account for the linkage signal. With the recent advancements in bioinformatics and molecular biology, QTL analysis combined with molecular-based strategies provides a systematic approach to identify candidate genes that contribute to various aspects of addictive behavior.