Urocortin is a novel neurotransmitter that appears to play a role in eating and drinking behavior. Most urocortin-positive (urocortin+) neurons in rodents are found in the cytoarchitecturally defined Edinger-Westphal nucleus (EW). However, the EW is traditionally described as the source of the preganglionic parasympathetic outflow to the ciliary ganglion. We examined the distribution of urocortin+ cells and motoneurons by use of immunohistochemical staining for this peptide and for choline acetyl transferase (ChAT) in macaque monkeys, where most preganglionic motoneurons inhabit the EW, and in cats, where most do not. In both species, lack of overt double labeling indicated the ChAT+ and urocortin+ cells are separate populations. In the monkey, most non-oculomotor ChAT+ neurons were found within the EW. In contrast, urocortin+ cells were mainly distributed between the oculomotor nuclei, and in the supraoculomotor area. In the cat, most non-oculomotor ChAT+ cells were located in the supraoculomotor area and anteromedian nucleus. Few were present in the cat EW. Instead, this nucleus was filled with urocortin+ cells. These results highlight the fact the term EW has come to indicate different nuclei in different species. Consequently, we have adopted the identifiers preganglionic (EWPG) and urocortin containing (EWU) to designate the cytoarchitecturally defined EW nuclei in monkeys and cats, respectively. Furthermore, we propose a new open-ended nomenclature for the perioculomotor (pIII) cells groups that have distinctive projections and neurochemical signatures. This will allow more effective scientific discourse on the connections and function of groups like the periculomotor urocortin (pIIIU) and preganglionic (pIIIPG) populations.

A common expression of neuroadaptations induced by repeated exposure to addictive drugs is a persistent sensitized behavioral response to their stimulant properties. Neuroplasticity underlying drug-induced sensitization has been proposed to explain compulsive drug pursuit and consumption characteristic of addiction. The hypothalamic-pituitary-adrenal (HPA) axis-activating neuropeptide, corticotropin-releasing factor (CRF), may be the keystone in drug-induced neuroadaptation. Corticosterone-activated glucocorticoid receptors (GRs) mediate the development of sensitization to ethanol (EtOH), implicating the HPA axis in this process. EtOH-induced increases in corticosterone require CRF activation of CRF1 receptors. We posited that CRF1 signaling pathways are crucial for EtOH-induced sensitization. We demonstrate that mice lacking CRF1 receptors do not show psychomotor sensitization to EtOH, a phenomenon that was also absent in CRF1 + 2 receptor double-knockout mice. Deletion of CRF2 receptors alone did not prevent sensitization. A blunted endocrine response to EtOH was found only in the genotypes showing no sensitization. The CRF1 receptor antagonist CP-154,526 attenuated the acquisition and prevented the expression of EtOH-induced psychomotor sensitization. Because CRF1 receptors are also activated by urocortin-1 (Ucn1), we tested Ucn1 knockout mice for EtOH sensitization and found normal sensitization in this genotype. Finally, we show that the GR antagonist mifepristone does not block the expression of EtOH sensitization. CRF and CRF1 receptors, therefore, are involved in the neurobiological adaptations that underlie the development and expression of psychomotor sensitization to EtOH. A CRF/CRF1-mediated mechanism involving the HPA axis is proposed for acquisition, whereas an extrahypothalamic CRF/CRF1 participation is suggested for expression of sensitization to EtOH.

There is evidence that the peptide urocortin 1 (Ucn1) may be involved in mediating some of the effects of ethanol. The purpose of the present study was to characterize Ucn1 immunoreactivity in mice selectively bred for either high or low sensitivity to ethanol-induced sedation, with additional differences in their response to ethanol-induced hypothermia. The brains of naïve male mice of the inbred long sleep/short sleep (ILS/ISS) selected lines were analyzed by immunohistochemistry. Significant differences were found between lines in the number of Ucn1-containing cells in the non-preganglionic Edinger-Westphal nucleus (npEW, the main source of Ucn1 in the brain); with the ISS mice having more cells. However, significant differences in the optical density of Ucn1 immunoreactivity in individual npEW cells and differences in cell area were also found between lines, with ILS mice having a greater density of Ucn1 per cell and having larger cells in the npEW. Importantly, the ILS mice also had a significantly greater number of Ucn1-positive terminal fibers than ISS mice in the lateral septum and the dorsal raphe nucleus, projection areas of Ucn1-containing neurons. These results suggest that the greater sensitivity of ILS than ISS mice to the hypothermic effects of ethanol could be mediated by stronger innervation of the dorsal raphe by Ucn1-containing fibers. In addition, these results lend further support to previous findings implicating Ucn1-containing projections from npEW to the dorsal raphe in ethanol-induced hypothermia.

The current study examined the effects of operant ethanol (EtOH) self-administration on gene expression kin the nucleus accumbens (ACB) and amygdala (AMYG) of inbred alcohol-preferring (iP) rats. Rats self-trained on a standard two-lever operant paradigm to administer either water-water, EtOH (15% v/v)-water, or saccharin (SAC; 0.0125% g/v)-water. Animals were killed 24 h after the last operant session, and the ACB and AMYG dissected; RNA was extracted and purified for microarray analysis. For the ACB, there were 513 significant differences at the p\textless0.01 level in named genes: 55 between SAC and water; 215 between EtOH and water, and 243 between EtOH and SAC. In the case of the AMYG (p\textless0.01), there were 48 between SAC and water, 23 between EtOH and water, and 63 between EtOH and SAC group. Gene Ontology (GO) analysis indicated that differences in the ACB between the EtOH and SAC groups could be grouped into 15 significant (p\textless0.05) categories, which included major categories such as synaptic transmission, cell and ion homeostasis, and neurogenesis, whereas differences between the EtOH and water groups had only 4 categories, which also included homeostasis and synaptic transmission. Several genes were in common between the EtOH and both the SAC and water groups in the synaptic transmission (e.g., Cav2, Nrxn3, Gabrb2, Gad1, Homer1) and homeostasis (S100b, Prkca, Ftl1) categories. Overall, the results suggest that changes in gene expression in the ACB of iP rats are associated with the reinforcing effects of EtOH.

The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high-throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.

Alcoholics generally display cycles of excessive ethanol intake, abstinence and relapse behavior. Using an animal model of relapse-like drinking, the alcohol deprivation effect (ADE), our laboratory has shown that repeated 2-week cycles of ethanol deprivation and re-exposure, following an initial 6 week access period, result in a robust ADE by alcohol-preferring (P) and high alcohol-drinking (HAD-1 and HAD-2) rats. These rat lines have been selectively bred to prefer a 10% ethanol solution over water. The present study examined whether P and HAD rats would display an ADE using much shorter ethanol deprivation and re-exposure intervals. Rats were given either continuous or periodic concurrent access to multiple concentrations [10%, 20%, and 30%, volume/volume (vol./vol.)] of ethanol. The periodic protocol involved access to ethanol for 12 days followed by 4 cycles of 4 days of deprivation and 4 days of re-exposure to ethanol access. HAD rats displayed a robust 24 hour ADE upon 1st re-exposure (HAD-1: \textasciitilde 5 vs. 8 g/kg/day; HAD-2: \textasciitilde 6 vs. 9 g/kg/day, baseline vs. re-exposure), whereas P rats (\textasciitilde 7 vs. 8 g/kg/day) displayed a modest, nonsignificant, increase in 24 hour intake. In a separate group of rats, ethanol intake and blood alcohol concentrations (BACs) after the 1st hour of the 4th re-exposure cycle were HAD-1: 2.0 g/kg and 97 mg%, HAD-2: 2.3 g/kg and 73 mg%, and P: 1.2 g/kg and 71 mg%; with all three lines displaying a robust 1st hour ADE. These findings suggest that (a) an ADE may be observed with short ethanol deprivation and re-exposure intervals in HAD rats, and (b) the genetic make-up of the P and HAD rats influences the expression of this ADE.

Many studies have used voluntary ethanol consumption by animals to assess the influence of genetic and environmental manipulations on ethanol drinking. However, the relationship between home cage ethanol consumption and more formal assessments of ethanol-reinforced behavior using operant and instrumental conditioning procedures is not always clear. The present review attempted to evaluate whether there are consistent correlations between mouse and rat home cage ethanol drinking on the one hand, and either operant oral self-administration (OSA), conditioned taste aversion (CTA), or conditioned place preference (CPP) with ethanol on the other. We also review literature on intravenous ethanol self-administration (IVSA). To collect data, we evaluated a range of genetic manipulations that can change both genes and ethanol drinking behavior including selective breeding, transgenic and knockout models, and inbred and recombinant inbred strain panels. For a genetic model to be included in the analysis, there had to be published data resulting in differences on home cage drinking and data for at least one of the other behavioral measures. A consistent, positive correlation was observed between ethanol drinking and OSA, suggesting that instrumental behavior is closely genetically related to consummatory and ingestive behavior directed at ethanol. A negative correlation was observed between CTA and drinking, suggesting that ethanol's aversive actions may limit oral consumption of ethanol. A more modest, positive relationship was observed between drinking and CPP, and there were not enough studies available to determine a relationship with IVSA. That some consistent outcomes were observed between widely disparate behavioral procedures and genetic populations may increase confidence in the validity of findings from these assays. These findings may also have important implications when researchers decide which phenotypes to use in measuring alcohol-reward relevant behaviors in novel animal models.

Background Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. At most central synapses, ethanol generally increases inhibitory synaptic transmission; however, no studies have explored the effect of acute ethanol on GABAergic transmission in the VTA. Methods Whole-cell patch clamp recordings of inhibitory postsynaptic currents (IPSCs) from VTA-DA neurons in midbrain slices from young rats. Results Acute exposure of VTA-DA neurons to ethanol (25 to 50 mM) robustly enhanced GABAergic spontaneous and miniature IPSC frequency while inducing a slight enhancement of spontaneous IPSC (sIPSC) amplitude. Ethanol (50 mM) enhanced paired-pulse depression of evoked IPSCs, further suggesting enhanced GABA release onto VTA-DA neurons. The frequency of sIPSCs was suppressed by the GABAB agonist, baclofen (1.25 μM) and enhanced by the antagonist, SCH50911 (20 μM); however, neither appeared to modulate or occlude the effects of ethanol on sIPSC frequency. Conclusions The present results indicate that ethanol increases postsynaptic GABAA receptor sensitivity, enhances action potential-independent GABA release onto VTA-DA neurons, and that this latter effect is independent of GABAB auto-receptor inhibition of GABA release.

The neurosteroid allopregnanolone (ALLO) is a positive modulator of GABAA receptors that exhibits a psychopharmacological profile similar to ethanol (i.e., anxiolytic, sedative-hypnotic). Based on research suggesting that manipulation of ALLO levels altered ethanol self-administration in male rodents, the current studies determined whether exogenous ALLO administration or the inhibition of its synthesis in vivo modulated ethanol intake patterns in female C57BL/6J mice. Lickometer circuits collected temporal lick records of ethanol (10% v/v) and water consumption during daily 2-hr limited access sessions. Following the establishment of stable ethanol intake, studies examined the effect of an acute ALLO challenge (3.2 – 24.0 mg/kg) or a 7-day blockade of ALLO production with finasteride (FIN; 50 or 100 mg/kg) on ethanol intake in a within-subjects design. In contrast to results in male mice, ethanol dose (g/kg), ethanol preference, and most of the bout parameters were unaltered by ALLO pretreatment in female mice. Ethanol intake in females also was recalcitrant to 7-day treatment with 50 mg/kg FIN, whereas 100 mg/kg FIN significantly reduced the ethanol dose consumed by 35%. The FIN-attenuated ethanol intake was attributable to a significant decrease in bout frequency (up to 45%), with lick patterns indicating reduced maintenance of consumption throughout the 2-hr session. FIN also produced a dose-dependent decrease in brain ALLO levels. In conjunction with data in male mice, the present findings indicate that there are sex differences in the physiological regulation of ethanol intake patterns by GABAergic neurosteroids.

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.