The role of withdrawal-related phenomena in the development and maintenance of alcohol addiction remains under debate. A 'self-medication' framework postulates that emotional changes are induced by a history of alcohol use, persist into abstinence, and are a major factor in maintaining alcoholism. This view initially focused on negative emotional states during early withdrawal: these are pronounced, occur in the vast majority of alcohol-dependent patients, and are characterized by depressed mood and elevated anxiety. This concept lost popularity with the realization that in most patients, these symptoms abate over 3-6 weeks of abstinence, while relapse risk persists long beyond this period. More recently, animal data have established that a prolonged history of alcohol dependence induces more subtle neuroadaptations. These confer altered emotional processing that persists long into protracted abstinence. The resulting behavioral phenotype is characterized by excessive voluntary alcohol intake and increased behavioral sensitivity to stress. Emerging human data support the clinical relevance of negative emotionality for protracted abstinence and relapse. These developments prompt a series of research questions: (1) are processes observed during acute withdrawal, while transient in nature, mechanistically related to those that remain during protracted abstinence?; (2) is susceptibility to negative emotionality in acute withdrawal in part due to heritable factors, similar to what animal models have indicated for susceptibility to physical aspects of withdrawal?; and (3) to what extent is susceptibility to negative affect that persists into protracted abstinence heritable?

Over the past 40 years, rigorous examination of brain function, structure, and attending factors through multidisciplinary research has helped identify the substrates of alcohol-related damage in the brain. One main area of this research has focused on the neuropsychological sequelae of alcoholism, which has resulted in the description of a pattern of sparing and impairment that provided an essential understanding of the functional deficits as well as of spared capabilities that could be useful in recovery. These studies have elucidated the component processes of memory, problem solving, and cognitive control, as well as visuospatial, and motor processes and their interactions with cognitive control processes. Another large area of research has focused on observable brain pathology, using increasingly sophisticated imaging technologies—progressing from pneumoencephalography to computed tomography, magnetic resonance imaging (MRI), diffusion tensor imaging, and functional MRI—that have enabled ever more detailed insight into brain structure and function. These advancements also have allowed analysis of the course of brain structural changes through periods of drinking, abstinence, and relapse.

Severe stress or trauma can cause permanent changes in brain circuitry, leading to dysregulation of fear responses and the development of posttraumatic stress disorder (PTSD). To date, little is known about the molecular mechanisms underlying stress-induced long-term plasticity in fear circuits. We addressed this question by using global gene expression profiling in an animal model of PTSD, stress-enhanced fear learning (SEFL). A total of 15 footshocks were used to induce SEFL and the volatile anesthetic isoflurane was used to suppress the behavioral effects of stress. Gene expression in lateral/basolateral amygdala was measured using microarrays at 3 weeks after the exposure to different combinations of shock and isoflurane. Shock produced robust effects on amygdalar transcriptome and isoflurane blocked or reversed many of the stress-induced changes. We used a modular approach to molecular profiles of shock and isoflurane and built a network of regulated genes, functional categories, and cell types that represent a mechanistic foundation of perturbation-induced plasticity in the amygdala. This analysis partitioned perturbation-induced changes in gene expression into neuron- and astrocyte-specific changes, highlighting a previously underappreciated role of astroglia in amygdalar plasticity. Many neuron-enriched genes were highly correlated with astrocyte-enriched genes, suggesting coordinated transcriptional responses to environmental challenges in these cell types. Several individual genes were validated using RT-PCR and behavioral pharmacology. This study is the first to propose specific cellular and molecular mechanisms underlying SEFL, an animal model of PTSD, and to nominate novel molecular and cellular targets with potential for therapeutic intervention in PTSD, including glycine and neuropeptide systems, chromatin remodeling, and gliotransmission.

C57BL/6J x FVB/NJ F1 (B6 x FVB) mice consume more alcohol than C57BL/6J x NZB/B1NJ F1 (B6 x NZB) mice and this high alcohol consumption is stable after abstinence whereas B6 x NZB show reduced consumption, thus providing models of Sustained Alcohol Preference (SAP) and Reduced Alcohol Preference (RAP). In female hybrids, we assessed several behavioral responses to define behaviors which might predict SAP and RAP. B6 x FVB exhibited less severe ethanol-induced conditioned taste aversion and were less sensitive to ethanol-induced loss of righting reflex than B6 x NZB. Both hybrids demonstrated ethanol-induced place preference and a low ethanol withdrawal severity. We found that these hybrids differ in their sensitivity to the aversive and sedative, but not rewarding, effects of ethanol. Results of elevated plus maze, mirror chamber, and locomotor tests reveal B6 x FVB mice are less anxious and more active than B6 x NZB mice. Results obtained offer insights about factors that determine SAP and RAP in these new genetic models of alcohol consumption.

Social factors have a tremendous influence on instances of heavy drinking and in turn impact public health. However, it is extremely difficult to assess whether this influence is only a cultural phenomenon or has biological underpinnings. Research in non-human primates demonstrates that the way individuals are brought up during early development affects their future predisposition for heavy drinking, and research in rats demonstrates that social isolation, crowding or low social ranking can lead to increased alcohol intake, while social defeat can decrease drinking. Neurotransmitter mechanisms contributing to these effects (i.e., serotonin, GABA, dopamine) have begun to be elucidated. However, these studies do not exclude the possibility that social effects on drinking occur through generalized stress responses to negative social environments. Alcohol intake can also be elevated in positive social situations, for example, in rats following an interaction with an intoxicated peer. Recent studies have also begun to adapt a new rodent species, the prairie vole, to study the role of social environment in alcohol drinking. Prairie voles demonstrate a high degree of social affiliation between individuals, and many of the neurochemical mechanisms involved in regulation of these social behaviors (for example, dopamine, central vasopressin and the corticotropin releasing factor system) are also known to be involved in regulation of alcohol intake. Naltrexone, an opioid receptor antagonist approved as a pharmacotherapy for alcoholic patients, has recently been shown to decrease both partner preference and alcohol preference in voles. These findings strongly suggest that mechanisms by which social factors influence drinking have biological roots, and can be studied using rapidly developing new animal models.

BACKGROUND: The binge-drinking model in rodents using intragastric injections of ethanol (EtOH) for 4 days results in argyrophilic corticolimbic tissue classically interpreted as indicating irreversible neuronal degeneration. However, recent findings suggest that acquired argyrophilia can also identify injured neurons that have the potential to recover. The current in vivo magnetic resonance (MR) imaging and spectroscopy study was conducted to test the hypothesis that binge EtOH exposure would injure but not cause the death of neurons as previously ascertained postmortem. METHODS: After baseline MR scanning, 11 of 19 rats received a loading dose of 5 g/kg EtOH via oral gavage, then a maximum of 3 g/kg every 8 hours for 4 days, for a total average cumulative EtOH dose of 43 +/- 1.2 g/kg and average blood alcohol levels of 258 +/- 12 mg/dL. All animals were scanned after 4 days of gavage (post-gavage scan) with EtOH (EtOH group) or dextrose (control [Con] group) and again after 7 days of abstinence from EtOH (recovery scan). RESULTS: Tissue shrinkage at the post-gavage scan was reflected by significantly increased lateral ventricular volume in the EtOH group compared with the Con group. At the post-gavage scan, the EtOH group had lower dorsal hippocampal N-acetylaspartate and total creatine and higher choline-containing compounds than the Con group. At the recovery scan, neither ventricular volume nor metabolite levels differentiated the groups. CONCLUSIONS: Rapid recovery of ventricular volume and metabolite levels with removal of the causative agent argues for transient rather than permanent effects of a single EtOH binge episode in rats.

Dynamic hyperpolarized [1-13C]pyruvate metabolic imaging in the normal anesthetized rat brain is demonstrated on a clinical 3-T magnetic resonance imaging scanner. A 12-second bolus injection of hyperpolarized [1-13C]pyruvate is imaged at a 3-second temporal resolution. The observed dynamics are evaluated with regard to cerebral blood volume (CBV), flow, transport, and metabolic exchange with the cerebral lactate pool. A model for brain [1-13C]lactate, based on blood–brain transport kinetics, CBV, and the observed pyruvate dynamics is described.

The objective of this study was to determine time-course changes in gene expression within two regions of the extended amygdala following binge-like alcohol drinking by alcohol-preferring (P) rats. Adult male P rats were given 1-hr access to 15 and 30% ethanol three times daily for 8 weeks. Rats (n = 10/time point for ethanol and n = 6/time point for water) were killed by decapitation 1, 6 and 24 hr after the last drinking episode. RNA was prepared from individual micropunch samples of the nucleus accumbens shell (ACB-shell) and central nucleus of the amygdala (CeA); analyses were conducted with Affymetrix Rat 230.2 chips. Ethanol intakes were 1.5–2 g/kg for each of the 3 sessions. There were no genes that were statistically different between the ethanol and water groups at any individual time point. Therefore, an overall effect, comparing the water and ethanol groups, was determined. In the ACB-shell and CeA, there were 276 and 402 probe sets for named genes, respectively, that differed between the two groups. There were 1.5- to 3.6- fold more genes with increased than decreased expression in the ethanol drinking group, with most differences between 1.1- to 1.2-fold. Among the differences between the ethanol and water groups were several significant Biological Processes categories that were in common between the 2 regions (e.g., synaptic transmission, neurite development); however, within these categories, there were few genes in common between the two regions. Overall, the results indicate that binge-like alcohol drinking by P rats produces region-dependent changes in the expression of genes that could alter transcription, synaptic function and neuronal plasticity in the ACB-shell and CeA; within each region, different mechanisms may underlie these alterations, since there were few common ethanol-responsive genes between the ACB-shell and CeA.

Evidence for genetic linkage to alcohol and other substance dependence phenotypes in areas of the human and mouse genome have now been reported with some consistency across studies. However, the question remains as to whether the genes that underlie the alcohol-related behaviors seen in mice are the same as those that underlie the behaviors observed in human alcoholics. The aims of the current set of analyses were to identify a small set of alcohol-related phenotypes in humans and in mouse by which to compare quantitative trait locus (QTL) data between the species using syntenic mapping. These analyses identified that QTLs for alcohol consumption and acute and chronic alcohol withdrawal on distal mouse chromosome 1 are syntenic to a region on human chromosome 1q where a number of studies have identified QTLs for alcohol-related phenotypes. Additionally, a QTL on human chromosome 15 for alcohol dependence severity/withdrawal identified in two human studies was found to be largely syntenic with a region on mouse chromosome 9 where two groups have found QTLs for alcohol preference. In both of these cases while the QTLs were found to be syntenic the exact phenotypes between humans and mice did not necessarily overlap. These studies demonstrate how this technique might be useful in the search for genes underlying alcohol-related phenotypes in multiple species. However, these findings also suggest that trying to match exact phenotypes in humans and mice may not be necessary or even optimal for determining whether similar genes influence a range of alcohol-related behaviors between the two species.

Risk for alcohol dependence in humans has substantial genetic contributions. Successful rodent models generally attempt to address only selected features of the human diagnosis. Most such models target the phenotype of oral administration of alcohol solutions, usually consumption of or preference for an alcohol solution versus water. Data from rats and mice for more than 50 years have shown genetic influences on preference drinking and related phenotypes. This paper summarizes some key findings from that extensive literature. Much has been learned, including the genomic location and possible identity of several genes influencing preference drinking. We report new information from congenic lines confirming QTLs for drinking on mouse chromosomes 2 and 9. There are many strengths of the various phenotypic assays used to study drinking, but there are also some weaknesses. One major weakness, the lack of drinking excessively enough to become intoxicated, has recently been addressed with a new genetic animal model, mouse lines selectively bred for their high and intoxicating blood alcohol levels after a limited period of drinking in the circadian dark. We report here results from a second replicate of that selection and compare them with the first replicate.

This review article discusses the importance of identifying gene-environment interactions for understanding the etiology and course of alcohol use disorders and related conditions. A number of critical challenges are discussed, including the fact that there is no organizing typology for classifying different types of environmental exposures, many key human environmental risk factors for alcohol dependence have no clear equivalents in other species, much of the genetic variance of alcohol dependence in human is not 'alcohol specific', and the potential range of gene-environment interactions that could be considered is so vast that maintaining statistical control of Type 1 errors is a daunting task. Despite these and other challenges, there appears to be a number of promising approaches that could be taken in order to achieve consilience and ecologically valid translation between human alcohol dependence and animal models. Foremost among these is to distinguish environmental exposures that are thought to have enduring effects on alcohol use motivation (and self-regulation) from situational environmental exposures that facilitate the expression of such motivations but do not, by themselves, have enduring effects. In order to enhance consilience, various domains of human approach motivation should be considered so that relevant environmental exposures can be sampled, as well as the appropriate species to study them in (i.e. where such motivations are ecologically relevant). Foremost among these are social environments, which are central to the initiation and escalation of human alcohol consumption. The value of twin studies, human laboratory studies and pharmacogenetic studies is also highlighted.

Background Corticotropin-releasing factor (CRF) and gamma-aminobutyric acid (GABA)ergic systems in the central amygdala (CeA) are implicated in the high-anxiety, high-drinking profile associated with ethanol dependence. Ethanol augments CeA GABA release in ethanol-naive rats and mice. Methods Using naive and ethanol-dependent rats, we compared electrophysiologic effects and interactions of CRF and ethanol on CeA GABAergic transmission, and we measured GABA dialyzate in CeA after injection of CRF1 antagonists and ethanol. We also compared mRNA expression in CeA for CRF and CRF1 using real-time polymerase chain reaction. We assessed effects of chronic treatment with a CRF1 antagonist on withdrawal-induced increases in alcohol consumption in dependent rats. Results CRF and ethanol augmented CeA GABAergic transmission in naive rats via increased GABA release. Three CRF1 receptor (CRF1) antagonists decreased basal GABAergic responses and abolished ethanol effects. Ethanol-dependent rats exhibited heightened sensitivity to CRF and CRF1 antagonists on CeA GABA release. Intra-CeA CRF1 antagonist administration reversed dependence–related elevations in GABA dialysate and blocked ethanol-induced increases in GABA dialyzate in both dependent and naive rats. Polymerase chain reaction studies indicate increased expression of CRF and CRF1 in CeA of dependent rats. Chronic CRF1 antagonist treatment blocked withdrawal-induced increases in alcohol drinking by dependent rats and tempered moderate increases in alcohol consumption by nondependent rats in intermittent testing. Conclusions These combined findings suggest a key role for specific presynaptic CRF-GABA interactions in CeA in the development and maintenance of ethanol dependence.

In every synapse, a large number of proteins interact with other proteins in order to carry out signaling and transmission in the central nervous system. In this study, we used interaction proteomics to identify novel synaptic protein interactions in mouse cortical membranes under native conditions. Using immunoprecipitation, immunoblotting, and mass spectrometry, we identified a number of novel synaptic protein interactions involving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), calcium-activated potassium channel (BKCa) alpha subunits, and dynamin-1. These novel interactions offer valuable insight into the protein-protein interaction network in intact synapses that could advance understanding of vesicle trafficking, release, and recycling.

The central amygdala (CeA) has a major role in alcohol dependence and reinforcement, and behavioral and neurochemical evidence suggests a role for the endocannabinoid (eCB) system in ethanol binging and dependence. We used a slice preparation to investigate the physiological role of cannabinoids and their interaction with ethanol on inhibitory synaptic transmission in CeA. Superfusion of the cannabinoid receptor (CB1) agonist WIN55212-2 (WIN2) onto CeA neurons decreased evoked GABAA receptor-mediated inhibitory postsynaptic potentials (IPSPs) in a concentration-dependent manner, an effect prevented by the CB1 antagonists Rimonabant (SR141716, SR1) and AM251. SR1 or AM251 applied alone augmented IPSPs, revealing a tonic eCB activity that decreased inhibitory transmission in CeA. Paired-pulse analysis suggested a presynaptic CB1 mechanism. Intracellular BAPTA abolished the ability of AM251 to augment IPSPs, demonstrating the eCB-driven nature and postsynaptic origin of the tonic CB1-dependent control of GABA release. Superfusion of ethanol increased IPSPs and addition of WIN2 reversed the ethanol effect. Similarly, previous superfusion of WIN2 prevented subsequent ethanol effects on GABAergic transmission. The ethanol-induced augmentation of IPSPs was additive to CB1 blockade, ruling out a participation of CB1 in the action of acute ethanol. Our study points to an important role of CB1 in CeA in which the eCBs tonically regulate neuronal activity, and suggests a potent mechanism for modulating CeA tone during challenge with ethanol.

The dopaminergic system originating in the midbrain ventral tegmental area (VTA) has been extensively studied over the past decades as a critical neural substrate involved in the development of alcoholism and addiction to other drugs of abuse. Accumulating evidence indicates that ethanol modulates the functional output of this system by directly affecting the firing activity of VTA dopamine neurons, whereas withdrawal from chronic ethanol exposure leads to a reduction in the functional output of these neurons. This chapter will provide an update on the mechanistic investigations of the acute ethanol action on dopamine neuron activity and the neuroadaptations/plasticities in the VTA produced by previous ethanol experience.

Dynamic membrane trafficking of the monoamine dopamine transporter (DAT) regulates dopaminergic signaling. Various intrinsic and pharmacological modulators can alter this trafficking. Previously we have shown ethanol potentiates in vitro DAT function and increases surface expression. However, the mechanism underlying these changes is unclear. In the present study, we found ethanol directly regulates DAT function by altering endosomal recycling of the transporter. We defined ethanol action on transporter regulation by [(3)H]DA uptake functional analysis combined with biochemical and immunological assays in stably expressing DAT HEK-293 cells. Short-term ethanol exposure potentiated DAT function in a concentration-, but not time-dependent manner. This potentiation was accompanied by a parallel increase in DAT surface expression. Ethanol had no effect on function or surface localization of the ethanol-insensitive mutant (G130T DAT), suggesting a trafficking-dependent mechanism in mediating the ethanol sensitivity of the transporter. The ethanol-induced increase in DAT surface expression occurred without altering the overall size of DAT endosomal recycling pools. We found ethanol increased the DAT membrane insertion rate while having no effect on internalization of the transporter. Ethanol had no effect on the surface expression or trafficking of the endogenously expressing transferrin receptor, suggesting ethanol does not have a nonspecific effect on endosomal recycling. These results define a novel trafficking mechanism by which ethanol regulates DAT function.

There is only modest overlap in the most common alcohol consumption phenotypes measured in animal studies and those typically studied in humans. To address this issue, we identified a number of alcohol consumption phenotypes of importance to the field that have potential for consilience between human and animal models. These phenotypes can be broken down into three categories: (1) abstinence/the decision to drink or abstain; (2) the actual amount of alcohol consumed; and (3) heavy drinking. A number of suggestions for human and animal researchers are made in order to address these phenotypes and enhance consilience. Laboratory studies of the decision to drink or to abstain are needed in both human and animal research. In human laboratory studies, heavy or binge drinking that meets cut-offs used in epidemiological and clinical studies should be reported. Greater attention to patterns of drinking over time is needed in both animal and human studies. Individual differences pertaining to all consumption phenotypes should be addressed in animal research. Lastly, improved biomarkers need to be developed in future research for use with both humans and animals. Greater precision in estimating blood alcohol levels in the field, together with consistent measurement of breath/blood alcohol levels in human laboratory and animal studies, provides one means of achieving greater consilience of alcohol consumption phenotypes.

The current study focused on the extent genetic diversity within a species (Mus musculus) affects gene co-expression network structure. To examine this issue, we have created a new mouse resource, a heterogeneous stock (HS) formed from the same eight inbred strains that have been used to create the collaborative cross (CC). The eight inbred strains capture \textgreater 90% of the genetic diversity available within the species. For contrast with the HS-CC, a C57BL/6J (B6) × DBA/2J (D2) F2 intercross and the HS4, derived from crossing the B6, D2, BALB/cJ and LP/J strains, were used. Brain (striatum) gene expression data were obtained using the Illumina Mouse WG 6.1 array, and the data sets were interrogated using a weighted gene co-expression network analysis (WGCNA).

Adaptations in the anterior cingulate cortex (ACC) have been implicated in alcohol and drug addiction. To identify genes that may contribute to excessive drinking, here we performed microarray analyses in laser microdissected rat ACC after a single or repeated administration of an intoxicating dose of alcohol (3g/kg). Expression of the small G protein K-ras was reduced following both single and repeated alcohol administration. We also observed that voluntary alcohol intake in K-ras heterozygous null mice (K-ras+/−) did not increased after withdrawal from repeated cycles of intermittent ethanol vapor exposure, unlike in their wild-type littermates. To identify K-ras regulated pathways, we then profiled gene expression in the ACC of K-ras+/−, heterozygous null mice for the K-ras negative regulator Nf1 (Nf1+/−) and wild-type mice following repeated administration of an intoxicating dose of alcohol. Pathway analysis showed that alcohol differentially affected various pathways in a K-ras dependent manner – some of which previously shown to be regulated by alcohol - including the insulin/PI3K pathway, the NF-kB, the phosphodiesterases (PDEs) pathway, the Jak/Stat and the adipokine signaling pathways. Altogether, the data implicate K-ras-regulated pathways in the regulation of excessive alcohol drinking after a history of dependence.