Ethanol increases dopaminergic release in the reward and reinforcement areas of the brain. The primary protein responsible for terminating dopamine (DA) neurotransmission is the plasma membrane-bound dopamine transporter (DAT). In vitro electrophysiological and biochemical studies in Xenopus laevis oocytes have previously shown ethanol potentiates DAT function and increases transporter-binding sites. The potentiating effect of ethanol on the transporter is eliminated in Xenopus oocytes by the DAT mutation glycine 130 to threonine. However, ethanol's action on DAT functional regulation has yet to be examined in mammalian cell expression systems. To further understand the molecular mechanisms of ethanol's action on DAT, we determined the direct mechanistic action of short-term (\textless or =2 h) ethanol exposure on transporter function and cell surface distribution in non-neuronal human embryonic kidney cells-293 (HEK-293) and neuronal SK-N-SH neuroblastoma cells expressing the transporter. Wild-type or G130T mutant DAT were overexpressed in HEK-293 and SK-N-SH cells. Ethanol potentiated DAT mediated [(3)H]DA uptake in a dose (25, 50, 100 mM), but not time dependent manner in cells expressing wild-type DAT. Ethanol-induced potentiation of uptake was significantly reduced in cells expressing the G130T mutant. Analysis of DA uptake kinetic parameters indicates 100-mM ethanol exposure increased [(3)H]DA uptake velocity (V(max)), while affinity for DA (K(m)) remained unchanged. The effect of ethanol on wild-type DAT surface expression was measured by biotinylation cell surface labeling. DAT surface expression increased 40%-50% after 1-h, 100-mM ethanol exposure. These studies show ethanol potentiates DAT functional regulation in both neuronal and non-neuronal cells, suggesting a direct mechanistic action of ethanol on transporter trafficking in mammalian systems. Our findings demonstrate ethanol's action on DAT function and regulation is consistent across multiple model systems.

Ethanol produces a wide variety of behavioral and physiological effects in the body, but exactly how it acts to produce these effects is still poorly understood. Although ethanol was long believed to act nonspecifically through the disordering of lipids in cell membranes, proteins are at the core of most current theories of its mechanisms of action. Although ethanol affects various biochemical processes such as neurotransmitter release, enzyme function, and ion channel kinetics, we are only beginning to understand the specific molecular sites to which ethanol molecules bind to produce these myriad effects. For most effects of ethanol characterized thus far, it is unknown whether the protein whose function is being studied actually binds ethanol, or if alcohol is instead binding to another protein that then indirectly affects the functioning of the protein being studied. In this Review, we describe criteria that should be considered when identifying alcohol binding sites and highlight a number of proteins for which there exists considerable molecular-level evidence for distinct ethanol binding sites.

There is substantial evidence that GABAergic neurotransmission is important for many behavioral actions of ethanol and there are reports spanning more than 30 years of literature showing that low to moderate (3–30 mM) concentrations of ethanol enhance GABAergic neurotransmission. A key question is which GABA receptor subunits are sensitive to low concentrations of ethanol in vivo and in vitro. Recent evidence points to a role for extrasynaptic receptors. Another question is which behavioral actions of alcohol result from enhancement of GABAergic neurotransmission. Some clues are beginning to emerge from studies of knock-out and knock-in mice and from genetic analysis of human alcoholics. These approaches are converging on a role for GABAergic actions in regulating alcohol consumption and, perhaps, the development of alcoholism.

The neuropeptide galanin and its three receptor subtypes (Gal R1-3) are highly expressed in the dorsal raphe nucleus (DRN), a region of the brain that contains a large population of serotonergic neurons. Galanin is co-expressed with serotonin in approximately 40% of the DRN neurons, and galanin and GALR2 expression are elevated by antidepressants like the SSRI fluoxetine, suggesting an interaction between serotonin and galanin. The present study examines the effect of galanin (Gal 1-29), a pan ligand for GalR (1-3) and the GalR2/GalR3-selective ligand, Gal 2-11, on the electrophysiological properties of DRN serotonergic neurons in a slice preparation. We recorded from cells in the DRN with electrophysiological characteristics consistent with those of serotonergic neurons that exhibit high input resistance, large after-hyperpolarizations and long spike duration as defined by Aghajanian and Vandermaelen. Both Gal 1-29 and Gal 2-11 decreased the amplitudes pharmacologically-isolated GABAergic inhibitory postsynaptic potentials (IPSPs) in these putative serotonergic neurons. Furthermore, based on paired pulse facilitation studies, we show that Gal 1-29 likely decreases GABA release through a presynaptic mechanism, whereas Gal 2-11 may act postsynaptically. These findings may enhance understanding of the cellular mechanisms underlying the effects of antidepressant treatments on galanin and galanin receptors in DRN.

The lateral hypothalamus (LH) is a brain structure that controls hedonic properties of both natural rewards and drugs of abuse. Mu opioid receptors are known to mediate drug reward, but whether overstimulation of these receptors impacts on LH function has not been studied. Here we have used a genome-wide microarray approach to identify LH responses to chronic mu opioid receptor activation at the transcriptional level. We have subjected wild-type and mu opioid receptor knockout mice to an escalating morphine regimen, which produces severe physical dependence in wild-type but not mutant animals. We have analyzed gene profiles in LH samples using the 430A.2 Affymetrix array and identified a set of 25 genes whose expression is altered by morphine in wild-type mice only. The regulation was confirmed for a subset of these genes using real-time quantitative PCR on samples from independent treatments. Altered expression of aquaporin 4, apolipoprotein D, and prostaglandin synthase is indicative of modified LH physiology. The regulation of two signaling genes (the serum glucocorticoid kinase and the regulator of G protein signaling 4) suggests that neurotransmission is altered in LH circuitry. Finally, the downregulation of apelin may indicate a potential role for this neuropeptide in opioid signaling and hedonic homeostasis. Altogether, our study shows that chronic mu opioid receptor stimulation induces gene expression plasticity in the LH and provides a unique collection of mu opioid receptor-dependent genes that potentially contribute to alter reward processes in addictive diseases.

Plentiful data from both animal and human studies support the importance of genetic influences in substance abuse and dependence (Bierut et al., 1998; Tsuang et al., 1998; Kendler et al., 2003). This review summarizes the evidence supporting such genetic influences, places them into perspective regarding animal and human studies, discusses the importance of both genes and environment, and highlights some specific genes of interest regarding the vulnerabilities for problems associated with alcohol use disorders. A long history of repetitive heavy use of alcohol exists across generations as well as the high prevalence of alcohol-related problems in Western societies. Moreover, the information offered here addresses the importance of more general issues regarding genetics and gene expression related to alcohol abuse and dependence.

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.

Alcohol "sensitivity" has been proposed as a predictive factor for development of alcohol dependence (Schuckit et al., 2005). Most measures of alcohol sensitivity in humans and animals include a component that can be ascribed to acute functional tolerance (AFT). AFT is a form of tolerance that develops within a single period of alcohol exposure and has a genetic component. We used microarray technology as well as quantitative trait locus analysis of phenotypic and gene expression data across 30 BXD recombinant inbred strains of mice, 20 inbred strains of mice, and two replicate lines of mice selectively bred for differences in AFT, to identify differentially expressed candidate genes that contribute to predisposition to AFT. Eight candidate genes were identified by our statistical and filtering methods. The location of brain expression of these genes was mapped using the Allen Brain Atlas (http://www.brain-map.org), and the transcript location and molecular pathway analysis indicated that brain structures and biochemical pathways implicated in long-term potentiation and memory might also participate in the generation of acute functional alcohol tolerance.

The Myc oncoprotein is a transcription factor involved in a variety of human cancers. Overexpression of Myc is associated with malignant transformation. In normal cells, Myc is induced by mitotic signals, and in turn, it regulates the expression of downstream target genes. Although diverse roles of Myc have been predicted from many previous studies, detailed functions of Myc targets are still unclear. By combining chromatin immunoprecipitation (ChIP) and promoter microarrays, we identified a total of 1469 Myc direct target genes, the majority of which are novel, in HeLa cells and human primary fibroblasts. We observed dramatic changes of Myc occupancy at its target promoters in foreskin fibroblasts in response to serum stimulation. Among the targets of Myc, 107 were nuclear encoded genes involved in mitochondrial biogenesis. Genes with important roles in mitochondrial replication and biogenesis, such as POLG, POLG2, and NRF1 were identified as direct targets of Myc, confirming a direct role for Myc in regulating mitochondrial biogenesis. Analysis of target promoter sequences revealed a strong preference for Myc occupancy at promoters containing one of several described consensus sequences, CACGTG, in vivo. This study thus sheds light on the transcriptional regulatory networks mediated by Myc in vivo.

Proteomics research is beginning to expand beyond the more traditional shotgun analysis of protein mixtures to include targeted analyses of specific proteins using mass spectrometry. Integral to the development of a robust assay based on targeted mass spectrometry is prior knowledge of which peptides provide an accurate and sensitive proxy of the originating gene product (i.e., proteotypic peptides). To develop a catalog of "proteotypic peptides" in human heart, TRIzol extracts of left-ventricular tissue from nonfailing and failing human heart explants were optimized for shotgun proteomic analysis using Multidimensional Protein Identification Technology (MudPIT). Ten replicate MudPIT analyses were performed on each tissue sample and resulted in the identification of 30 605 unique peptides with a q-value \textless or = 0.01, corresponding to 7138 unique human heart proteins. Experimental observation frequencies were assessed and used to select over 4476 proteotypic peptides for 2558 heart proteins. This human cardiac data set can serve as a public reference to guide the selection of proteotypic peptides for future targeted mass spectrometry experiments monitoring potential protein biomarkers of human heart diseases.

Drug addiction is a chronic, relapsing disorder, characterized by an uncontrollable motivation to seek and use drugs. Converging clinical and preclinical observations implicate pathologies within the corticolimbic glutamate system in the genetic predisposition to, and the development of, an addicted phenotype. Such observations pose cellular factors regulating glutamate transmission as likely molecular candidates in the etiology of addiction. Members of the Homer family of proteins regulate signal transduction through, and the trafficking of, glutamate receptors, as well as maintain and regulate extracellular glutamate levels in corticolimbic brain regions. This review summarizes the existing data implicating the Homer family of protein in acute behavioral and neurochemical sensitivity to drugs of abuse, the development of drug-induced neuroplasticity, as well as other behavioral and cognitive pathologies associated with an addicted state.

Background:  Allopregnanolone (ALLO) is a physiologically relevant neurosteroid modulator of GABAA receptors, and it exhibits a psychopharmacological profile that closely resembles the post-ingestive effects of ethanol. The 5α-reductase inhibitor finasteride (FIN), which inhibits biosynthesis of ALLO and structurally related neurosteroids, was previously demonstrated to reduce the maintenance of limited-access ethanol consumption. The primary aim of the current work was to determine whether FIN would reduce the acquisition of drinking in ethanol-naïve mice. Methods:  Male C57BL/6J (B6) mice were acclimated to a reverse light/dark schedule, and were provided ad libitum access to chow and water. Following habituation to vehicle injections (VEH; 20% w/v β-cyclodextrin; i.p.) administered 22-hour prior to drinking sessions with water only, mice were divided into 3 treatment groups: vehicle control (VEH), 50 mg/kg FIN (FIN-50), and 100 mg/kg FIN (FIN-100). Twenty-two hours after the first treatment, mice were permitted the inaugural 2-hour limited access to a 10% v/v ethanol solution (10E) and water. The acquisition of 10E consumption and underlying drinking patterns were assessed during FIN treatment (7 days) and subsequent FIN withdrawal (13 days) phases. Results:  FIN dose-dependently blocked the acquisition of 10E drinking and prevented the development of ethanol preference, thereby suggesting that the GABAergic neurosteroids may be important in the establishment of stable drinking patterns. FIN-elicited reductions in 10E intake were primarily attributable to selective and marked reductions in bout frequency, as no changes were observed in bout size, duration, or lick rates following FIN treatment. FIN-treated mice continued to exhibit attenuated ethanol consumption after 2 weeks post-treatment, despite a full recovery in brain ALLO levels. A second study confirmed the rightward and downward shift in the acquisition of ethanol intake following 7 daily FIN injections. While there were no significant group differences in brain ALLO levels following the seventh day of ethanol drinking, ALLO levels were decreased by 28% in the FIN-50 group. Conclusions:  Although the exact mechanism is unclear, FIN and other pharmacological interventions that modulate the GABAergic system may prove useful in curbing ethanol intake acquisition in at-risk individuals.

Microarray analyses of human MDA-MB-435 breast cancer cells treated with vitamin E analog 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl) chroman-6-yloxy acetic acid (α-TEA) showed over 400 genes to be modulated. Thirty-four genes deemed of interest based on potential involvement in anticancer activities of α-TEA fell into six categories: apoptosis related, signal transduction, cell cycle related, cell adhesion and motility, transcriptional regulators, and membrane traffic related. The gene (PMAIP1) for NOXA was studied further. NOXA mRNA and protein levels were elevated in a time and dose-dependent fashion following α-TEA treatment. Functional knockdowns using small interfering RNA (siRNA) showed NOXA to contribute to α-TEA-induced apoptosis. A correlation between α-TEA's ability to upregulate NOXA and induce apoptosis was seen among several human breast cancer cell lines. Efforts to identify upstream regulators of NOXA in α-TEA-induced apoptosis identified the necessity of both c-Jun N-terminal kinase (JNK) activation and p73 expression. Additionally, protein levels of full length p73 were decreased by JNK siRNA treatment, suggesting that the signal transduction module of JNK-p73-NOXA is involved in α-TEA induced apoptosis of human breast cancer cells. Taken together, these findings suggest a role for JNK activation in mediating full length p73 expression and add to our understanding of the mechanisms of anticancer actions of α-TEA, a potential chemotherapeutic agent. © 2007 Wiley-Liss, Inc.

Physiological dependence and associated withdrawal episodes are thought to constitute a motivational force that sustains ethanol (alcohol) use/abuse and may contribute to relapse in alcoholics. Although no animal model duplicates alcoholism, models for specific factors, like the withdrawal syndrome, are useful for identifying potential genetic and neural determinants of liability in humans. We generated congenic mice that confirm a quantitative trait locus (QTL) on chromosome 4 with a large effect on predisposition to alcohol withdrawal. Using c-Fos expression as a high-resolution marker of neuronal activation, congenic mice demonstrated significantly less neuronal activity associated with ethanol withdrawal than background strain mice in the substantia nigra pars reticulata (SNr), subthalamic nucleus (STN), rostromedial lateral globus pallidus, and ventral pallidum. Notably, neuronal activation in subregions of the basal ganglia associated with limbic function was more intense than in subregions associated with sensorimotor function. Bilateral lesions of caudolateral SNr attenuated withdrawal severity after acute and repeated ethanol exposures, whereas rostrolateral SNr and STN lesions did not reduce ethanol withdrawal severity. Caudolateral SNr lesions did not affect pentylenetetrazol-enhanced convulsions. Our results suggest that this QTL impacts ethanol withdrawal via basal ganglia circuitry associated with limbic function and that the caudolateral SNr plays a critical role. These are the first analyses to elucidate circuitry by which a confirmed addiction-relevant QTL influences behavior. This mouse QTL is syntenic with human chromosome 9p. Given the growing body of evidence that a gene(s) on chromosome 9p influences alcoholism, our results can facilitate human research on alcohol dependence and withdrawal.

Label-free relative quantitative proteomics is a powerful tool for the survey of protein level changes between two biological samples. We have developed and applied an algorithm using chromatographic alignment of μLC−MS runs to improve the detection of differences between complex protein mixtures. We demonstrate the performance of our software by finding differences in E. coli protein abundance upon induction of the lac operon genes using isopropyl β-d-thiogalactopyranoside. The use of our alignment gave a 4-fold decrease in mean relative retention time error and a 6-fold increase in the number of statistically significant differences between samples. Using a conservative threshold, we have identified 5290 total μLC−MS regions that have a different abundance between these samples. Of the detected difference regions, only 23% were mapped to MS/MS peptide identifications. We detected 74 proteins that had a greater relative abundance in the induced sample and 21 with a greater abundance in the uninduced sample. We have developed an effective tool for the label-free detection of differences between samples and demonstrate an increased sensitivity following chromatographic alignment.

BACKGROUND: The central extended amygdala (cEA) which includes the central nucleus of the amygdala (CeA) and the lateral posterior bed nucleus of the stria terminalis (BNSTLP), has been proposed to play a key role in excessive ethanol consumption in humans (Koob and Le Moal, 2005 Nat Neurosci 8:1442). To examine this relationship, we used a murine model of ethanol dependence (Becker and Lopez, 2004 Alcohol Clin Exp Res 28:1829; Lopez and Becker, 2005 Psychopharmacology (Berl) 181:688) and compared animals with sham lesions and electrolytic lesions of the CeA and BNSTLP. METHODS: Male C57BL/6J (B6) mice were first acclimated to a limited-access 2-bottle-choice preference procedure. The access period began 3 hours into the dark phase of the light-dark cycle and continued for 2 hours. Once acclimated (1 week), mice underwent chronic exposure to and intermittent withdrawal from ethanol vapor. The animals were then retested in the limited-access 2-bottle-choice preference procedure. In some experiments, electrolytic and sham lesions of the CeA or BNSTLP were performed prior to initiating the 2-bottle choice procedure. RESULTS: In a series of 5 preliminary experiments, mice were randomly assigned either to the standard intermittent ethanol vapor procedure or to the standard procedure but with air in the vapor chamber (control). The air-control procedure produced no change in ethanol intake when compared to baseline consumption. In contrast, intermittent ethanol vapor exposure increased ethanol consumption by almost 50%. The increase in consumption was associated with an increase in total fluid volume consumed and no change in ethanol preference. Lesions of both the BNSTLP and CeA significantly decreased baseline ethanol consumption, the former by decreasing fluid consumption and the latter by decreasing ethanol preference. Intermittent ethanol vapor exposure significantly increased consumption in both the BNSTLP- and CeA-lesioned animals, largely by increasing the total volume of fluid consumed. CONCLUSIONS: The results obtained clearly demonstrate that the cEA has a role in the regulation of ethanol consumption in the limited-access procedure. However, neither lesions of the CeA nor BNSTLP prevented the intermittent ethanol vapor-induced increase in consumption. These data do not preclude some role of the cEA in the increased ethanol consumption following intermittent ethanol vapor exposure, but would suggest that other brain regions also must have a significant influence.

Magnetic resonance imaging (MRI) provides a safe, noninvasive method to examine the brain’s macrostructure, microstructure, and some aspects of how the living brain functions. MRI is capable of detecting abnormalities that can occur with alcoholism as well as changes that can occur with sobriety and relapse. The brain pathology associated with chronic excessive alcohol consumption is well documented with imaging of the living body (i.e., in vivo imaging). Consistent findings include shrinkage of the frontal cortex,1 underlying white matter, and cerebellum and expansion of the ventricles. Some of these changes are reversible with abstinence, but some appear to be enduring. Research showing correlations between brain structure and quantitative neuropsychological testing demonstrates the functional consequences of the pathology. In addition, functional imaging studies provide evidence that the brain compensates for cognitive deficits. The myriad concomitants of alcoholism, the antecedents, and the consumption patterns each may influence the observed brain changes associated with alcoholism, which tend to be more deleterious with increasing age. The multifaceted nature of alcoholism presents unique challenges and opportunities to understand the mechanisms underlying alcoholism-induced neuropathology and its recovery. Longitudinal MRI studies of animal models of alcoholism, however, can address questions about the development and course of alcohol dependence and the scope and limits of in vivo degeneration and recovery of brain structure and concomitant function that may not be readily addressed in clinical studies.

Recent studies have demonstrated that metabotropic glutamate receptor 5 (mGluR5) antagonists decrease alcohol self-administration and suggest that the anti-craving medication, acamprosate, may also act to decrease mGluR5 function. To address the role of mGluR5 in behavioural actions of ethanol and acamprosate, we compared mutant mice with deletion of the mGluR5 gene and mice treated with a mGluR5 antagonist (MPEP) or acamprosate. Lack of mGluR5 or administration of MPEP reduced the severity of alcohol-induced withdrawal (AW), increased the sedative effect of alcohol (duration of loss of righting reflex; LORR), and increased basal motor activity. The motor stimulation produced by ethanol was blocked by deletion of mGluR5, but not by injection of MPEP. Both acamprosate and MPEP increased ethanol-induced LORR and reduced AW. Importantly, the protective effects of both MPEP and acamprosate on AW were found when the drugs were injected before, but not after, injection of ethanol. This indicates that the drugs prevented development of dependence rather than merely producing an anticonvulsant action. No effects of acamprosate or MPEP on ethanol-induced LORR and AW were found in mGluR5 knockout mice, demonstrating that mGluR5 is required for these actions. mGluR5 null mutant mice showed decreased alcohol consumption in some, but not all, tests. These data show the importance of mGluR5 for several actions of alcohol and support the hypothesis that some effects of acamprosate require mGluR5 signalling.

Addiction develops from the gradual adaptation of the brain to chronic drug exposure, and involves genetic reprogramming of neuronal function. The central extended amygdala (EAc) is a network formed by the central amygdala and the bed nucleus of the stria terminalis. This key site controls drug craving and seeking behaviors, and has not been investigated at the gene regulation level. We used Affymetrix microarrays to analyze transcriptional activity in the murine EAc, with a focus on mu-opioid receptor-associated events because these receptors mediate drug reward and dependence. We identified 132 genes whose expression is regulated by a chronic escalating morphine regimen in the EAc from wild-type but not mu-opioid receptor knockout mice. These modifications are mostly EAc-specific. Gene ontology analysis reveals an overrepresentation of neurogenesis, cell growth and signaling protein categories. A separate quantitative PCR analysis of genes in the last of these groups confirms the dysregulation of both orphan (Gpr88) and known (DrD1A, Adora2A, Cnr1, Grm5, Gpr6) G protein-coupled receptors, scaffolding (PSD95, Homer1) and signaling (Sgk, Cap1) proteins, and neuropeptides (CCK, galanin). These transcriptional modifications do not occur following a single morphine injection, and hence result from long-term adaptation to excessive mu receptor activation. Proteins encoded by these genes are classically associated with spine modules function in other brain areas, and therefore our data suggest a remodeling of EAc circuits at sites where glutamatergic and monoaminergic afferences interact. Together, mu receptor-dependent genes identified in this study potentially contribute to drug-induced neural plasticity, and provide a unique molecular repertoire towards understanding drug craving and relapse.