BACKGROUND: The current therapies for alcohol abuse disorders are not effective in all patients, and continued development of pharmacotherapies is needed. One approach that has generated recent interest is the antagonism of ghrelin receptors. Ghrelin is a gut-derived peptide important in energy homeostasis and regulation of hunger. Recent studies have implicated ghrelin in alcoholism, showing altered plasma ghrelin levels in alcoholic patients as well as reduced intakes of alcohol in ghrelin receptor knockout mice and in mice treated with ghrelin receptor antagonists. The aim of this study was to determine the neuroanatomical locus/loci of the effect of ghrelin receptor antagonism on alcohol consumption using the ghrelin receptor antagonist, D-Lys3-GHRP-6. METHODS: In Experiment 1, male C57BL/6J mice were injected with saline 3 hours into the dark cycle and allowed access to 15% (v/v) ethanol or water for 2 hours in a 2-bottle choice experiment. On test day, the mice were injected with either saline or 400 nmol of the ghrelin receptor antagonist, D-Lys3-GHRP-6, and allowed to drink 15% ethanol or water for 4 hours. The preference for alcohol and alcohol intake were determined. In Experiment 2, the same procedure was followed as in Experiment 1 but mice were only allowed access to a single bottle of 20% ethanol (v/v), and alcohol intake was determined. Blood ethanol levels were analyzed, and immunohistochemistry for c-Fos was carried out to investigate changes in neural activity. To further elucidate the mechanism by which D-Lys3-GHRP-6 affects alcohol intake, in Experiment 3, the effect of D-Lys3-GHRP-6 on the neural activation induced by intraperitoneal ethanol was investigated. For the c-Fos studies, brain regions containing ghrelin receptors were analyzed, i.e. the perioculomotor urocortin population of neurons (pIIIu), the ventral tegmental area (VTA), and the arcuate nucleus (Arc). In Experiment 4, to test if blood ethanol concentrations were affected by D-Lys3-GHRP-6, blood samples were taken at 2 time-points after D-Lys3-GHRP-6 pretreatment and systemic ethanol administration. RESULTS: In Experiment 1, D-Lys3-GHRP-6 reduced preference to alcohol and in a follow-up experiment (Experiment 2) also dramatically reduced alcohol intake when compared to saline-treated mice. The resulting blood ethanol concentrations were lower in mice treated with the ghrelin receptor antagonist. Immunohistochemistry for c-Fos showed fewer immunopositive cells in the pIIIu of the antagonist-treated mice but no difference was seen in the VTA or Arc. In Experiment 3, D-Lys3-GHRP-6 reduced the induction of c-Fos by intraperitoneal ethanol in the pIIIu but had no effect in the VTA. In the Arc, there was a significant increase in the number of c-Fos immunopositive cells after D-Lys3-GHRP-6 administration, but the antagonist had no effect on ethanol-induced expression of c-Fos. D-Lys3-GHRP-6-pretreatment also did not affect the blood ethanol concentrations observed after a systemic injection of ethanol when compared to saline-pretreated mice (Experiment 4). CONCLUSIONS: These findings indicate that the action of ghrelin on the regulation of alcohol consumption may occur via the pIIIu.

If people are brought into the laboratory and given alcohol, there are pronounced differences among individuals in many responses to the drug. Some participants in alcohol challenge protocols show a cluster of 'low level of responses to alcohol' determined by observing post-drinking-related changes in subjective, motor and physiological effects at a given dose level. Those individuals characterized as having low level of response (LR) to alcohol have been shown to be at increased risk for a lifetime diagnosis of alcohol dependence (AD), and this relationship between low LR and AD appears to be in part genetic. LR to alcohol is an area where achieving greater consilience between the human and the rodent phenotypes would seem to be highly likely. However, despite extensive data from both human and rodent studies, few attempts have been made to evaluate the human and animal data systematically in order to understand which aspects of LR appear to be most directly comparable across species and thus the most promising for further study. We review four general aspects of LR that could be compared between humans and laboratory animals: (1) behavioral measures of subjective intoxication; (2) body sway; (3) endocrine responses; and (4) stimulant, autonomic and electrophysiological responses. None of these aspects of LR provide completely face-valid direct comparisons across species. Nevertheless, one of the most replicated findings in humans is the low subjective response, but, as it may reflect either aversively valenced and/or positively valenced responses to alcohol as usually assessed, it is unclear which rodent responses are analogous. Stimulated heart rate appears to be consistent in animal and human studies, although at-risk subjects appear to be more rather than less sensitive to alcohol using this measure. The hormone and electrophysiological data offer strong possibilities of understanding the neurobiological mechanisms, but the rodent data in particular are rather sparse and unsystematic. Therefore, we suggest that more effort is still needed to collect data using refined measures designed to be more directly comparable in humans and animals. Additionally, the genetically mediated mechanisms underlying this endophenotype need to be characterized further across species.

We showed that F1 hybrid genotypes may provide a broader variety of ethanol drinking phenotypes than the inbred progenitor strains used to create the hybrids (Blednov et al. in Alcohol Clin Exp Res 29:1949–1958–2005). To extend this work, we characterized alcohol consumption as well as intake of other tastants (saccharin, quinine and sodium chloride) in five inbred strains of mice (FVB, SJL, B6, BUB, NZB) and in their reciprocal F1 hybrids with B6 (FVBxB6; B6xFVB; NZBxB6; B6xNZB; BUBxB6; B6xBUB; SJLxB6; B6xSJL). We also compared ethanol intake in these mice for several concentrations before and after two periods of abstinence. F1 hybrid mice derived from the crosses of B6 and FVB and also B6 and SJL drank higher levels of ethanol than their progenitor strains, demonstrating overdominance for two-bottle choice drinking test. The B6 and NZB hybrid showed additivity in two-bottle choice drinking, whereas the hybrid of B6 and BUB demonstrated full or complete dominance. Genealogical origin, as well as non-alcohol taste preferences (sodium chloride), predicted ethanol consumption. Mice derived from the crosses of B6 and FVB showed high sustained alcohol preference and the B6 and NZB hybrids showed reduced alcohol preference after periods of abstinence. These new genetic models offer some advantages over inbred strains because they provide high, sustained, alcohol intake, and should allow mapping of loci important for the genetic architecture of these traits.

A fast spiral chemical shift imaging (CSI) has been developed to address the challenge of the limited acquisition window in hyperpolarized (13)C metabolic imaging. The sequence exploits the sparsity of the spectra and prior knowledge of resonance frequencies to reduce the measurement time by undersampling the data in the spectral domain. As a consequence, multiple reconstructions are necessary for any given data set as only frequency components within a selected bandwidth are reconstructed "in-focus" while components outside that band are severely blurred ("spectral tomosynthesis"). A variable-flip-angle scheme was used for optimal use of the longitudinal magnetization. The sequence was applied to sub-second metabolic imaging of the rat in vivo after injection of hyperpolarized [1-(13)C]-pyruvate on a clinical 3T MR scanner. The comparison with conventional CSI based on phase encoding showed similar signal-to-noise ratio (SNR) and spatial resolution in metabolic maps for the substrate and its metabolic products lactate, alanine, and bicarbonate, despite a 50-fold reduction in scan time for the spiral CSI acquisition. The presented results demonstrate that dramatic reductions in scan time are feasible in hyperpolarized (13)C metabolic imaging without a penalty in SNR or spatial resolution.

BACKGROUND: Gamma-aminobutyric acid type B (GABAB) receptors decrease neural activity through G protein signaling. There are two subunits, GABAB1 and GABAB2. Alternative splicing provides GABAB1 with structural and functional diversity. cDNA microarrays showed strong signals from human brain RNA using GABAB1 intron 4 region probes. Therefore, we predicted the existence of novel splice variants. METHODOLOGY/PRINCIPAL FINDINGS: Based on the probe sequence analysis, we proposed two possible splice variants, GABAB1j and GABAB1k. The existence of human GABAB1j was verified by quantitative real-time PCR, and mouse GABAB1j was found from a microarray probe set based on human GABAB1j sequence. GABAB1j open reading frames (ORF) and expression patterns are not conserved across species, and they do not have any important functional domains except sushi domains. Thus, we focused on another possible splice variant, GABAB1k. After obtaining PCR evidence for GABAB1k existence from human, mouse, and rat, it was cloned from human and mouse by PCR along with three additional isoforms, GABAB1l, GABAB1m, and GABAB1n. Their expression levels by quantitative real-time PCR are relatively low in brain although they may be expressed in specific cell types. GABAB1l and GABAB1m inhibit GABAB receptor-induced G protein-activated inwardly rectifying K(+) channel (GIRK) currents at Xenopus oocyte two-electrode voltage clamp system. CONCLUSIONS/SIGNIFICANCE: This study supports previous suggestions that intron 4 of GABAB1 gene is a frequent splicing spot across species. Like GABAB1e, GABAB1l and GABAB1m do not have transmembrane domains but have a dimerization motif. So, they also could be secreted and bind GABAB2 dominantly instead of GABAB1a. However, only GABAB1l and GABAB1m are N- and C-terminal truncated splicing variants and impair receptor function. This suggests that the intron 4 containing N-terminal truncation is necessary for the inhibitory action of the new splice variants.

BACKGROUND: In rodent and human studies, ethanol (EtOH) exposure is associated with elevated brain levels of the magnetic resonance spectroscopy (MRS) signal representing choline-containing compounds (Cho). One interpretation of elevated brain Cho is that it is a marker of neuroinflammation, and some evidence suggests that EtOH exposure promotes neuroinflammation. This study aimed to determine whether binge EtOH exposure (intragastric 3 g/kg 25% EtOH every 8 hours for 4 days) would induce the expression of certain cytokines in blood, liver, or brain, thereby supporting the neuroinflammation hypothesis of elevated Cho. METHODS: Ten of 18 wild-type male Wistar rats (\textasciitilde322 g at baseline) were exposed to EtOH and attained average blood alcohol levels of \textasciitilde315 mg/dl across 4 days. Blood for cytokine immunoassays was collected at baseline, after 5 doses of EtOH (binge), and immediately preceding euthanasia either 4 or 24 hours after the last dose of EtOH. Blood was additionally assayed for the levels of thiamine and liver enzymes; liver histopathology was performed postmortem; and tissue from liver and 6 brain regions was assayed for the potential induction of 7 cytokines. RESULTS: There were no group effects on the levels of thiamine or its phosphate derivatives, thiamine monophosphate or thiamine diphosphate. ANOVAs of liver enzyme levels indicated that only alkaline phosphatase (ALP) levels were higher in the EtOH group than in control group at binge; ALP elevations, however, are difficult to explain in the absence of changes in the levels of additional liver enzymes. Postmortem liver pathology provided evidence for minimal microvesicular lipidosis and portocentric fibrosis in the EtOH group. Group effects on the levels of the measured cytokines in the blood (TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-13, and GRO/CXCL1) were not significant. Similarly, postmortem evaluation of liver cytokines did not reveal group effects. Postmortem evaluation of the 7 cytokines in 6 brain regions (anterior cerebellar vermis, cingulate cortex, frontal cortex, hippocampus, hypothalamus, striatum) also failed to identify group effects. CONCLUSIONS: A single 4-day bout of binge EtOH exposure alone was insufficient to induce the expression of 7 cytokines in blood, liver, or 6 brain regions of wild-type Wistar rats. Alternative interpretations for elevations in brain Cho in response to a 4-day binge EtOH treatment are therefore necessary and may include induction of cytokines not measured herein or other noninflammatory mechanisms.

Formulation, polarization, and dissolution conditions were developed to obtain a stable hyperpolarized solution of [1-(13)C]-ethyl pyruvate. A maximum tolerated concentration and injection rate were determined, and (13)C spectroscopic imaging was used to compare the uptake of hyperpolarized [1-(13)C]-ethyl pyruvate relative to hyperpolarized [1-(13)C]-pyruvate into anesthetized rat brain. Hyperpolarized [1-(13)C]-ethyl pyruvate and [1-(13)C]-pyruvate metabolic imaging in normal brain is demonstrated and quantified in this feasibility and range-finding study.

Excessive alcohol (ethanol) consumption is the hallmark of alcohol use disorders. The F1 hybrid cross between the C57BL/6J (B6) and FVB/NJ (FVB) inbred mouse strains consumes more ethanol than either progenitor strain. The purpose of this study was to utilize ethanol-drinking data and genetic information to map genes that result in overdominant (or heterotic) ethanol drinking. About 600 B6 x FVB F2 mice, half of each sex, were tested for ethanol intake and preference in a 24-h, two-bottle water versus ethanol choice procedure, with ascending ethanol concentrations. They were then tested for ethanol intake in a Drinking in the Dark (DID) procedure, first when there was no water choice and then when ethanol was offered versus water. DNA samples were obtained and genome-wide QTL analyses were performed to search for single QTLs (both additive and dominance effects) and interactions between pairs of QTLs, or epistasis. On average, F2 mice consumed excessive amounts of ethanol in the 24-h choice procedure, consistent with high levels of consumption seen in the F1 cross. Consumption in the DID procedure was similar or higher than amounts reported previously for the B6 progenitor. QTLs resulting in heightened consumption in heterozygous compared to homozygous animals were found on Chrs 11, 15, and 16 for 24-h choice 30% ethanol consumption, and on Chr 11 for DID. No evidence was found for epistasis between any pair of significant or suggestive QTLs. This indicates that the hybrid overdominance is due to intralocus interactions at the level of individual QTL.

Gamma-aminobutyric acid (GABA) is synthesized in brain by two isoforms of glutamic acid decarboxylase (Gad), Gad1 and Gad2. Gad1 provides most of the GABA in brain, but Gad2 can be rapidly activated in times of high GABA demand. Mice lacking Gad2 are viable whereas deletion of Gad1 is lethal. We produced null mutant mice for Gad2 on three different genetic backgrounds: predominantly C57BL/6J and one or two generations of backcrossing to 129S1/SvimJ (129N1, 129N2). We used these mice to determine if actions of alcohol are regulated by synthesis of GABA from this isoform. We also studied behavioral responses to a benzodiazepine (flurazepam) and a GABAA receptor agonist (gabaxadol). Deletion of Gad2 increased ethanol palatability and intake and slightly reduced the severity of ethanol-induced withdrawal, but these effects depended strongly on genetic background. Mutant mice on the 129N2 background showed the above three ethanol behavioral phenotypes, but the C57BL/6J inbred background did not show any of these phenotypes. Effects on ethanol consumption also depended on the test as the mutation did not alter consumption in limited access models. Deletion of Gad2 reduced the effect of flurazepam on motor incoordination and increased the effect of extrasynaptic GABAA receptor agonist gabaxadol without changing the duration of loss of righting reflex produced by these drugs. These results are consistent with earlier proposals that deletion of Gad2 (on 129N2 background) reduces synaptic GABA but also suggest changes in extrasynaptic receptor function.

Gamma-aminobutyric acid (GABA) is synthesized in brain by two isoforms of glutamic acid decarboxylase (Gad), Gad1 and Gad2. Gad1 provides most of the GABA in brain, but Gad2 can be rapidly activated in times of high GABA demand. Mice lacking Gad2 are viable whereas deletion of Gad1 is lethal. We produced null mutant mice for Gad2 on three different genetic backgrounds: predominantly C57BL/6J and one or two generations of backcrossing to 129S1/SvimJ (129N1, 129N2). We used these mice to determine if actions of alcohol are regulated by synthesis of GABA from this isoform. We also studied behavioral responses to a benzodiazepine (flurazepam) and a GABAA receptor agonist (gabaxadol). Deletion of Gad2 increased ethanol palatability and intake and slightly reduced the severity of ethanol-induced withdrawal, but these effects depended strongly on genetic background. Mutant mice on the 129N2 background showed the above three ethanol behavioral phenotypes, but the C57BL/6J inbred background did not show any of these phenotypes. Effects on ethanol consumption also depended on the test as the mutation did not alter consumption in limited access models. Deletion of Gad2 reduced the effect of flurazepam on motor incoordination and increased the effect of extrasynaptic GABAA receptor agonist gabaxadol without changing the duration of loss of righting reflex produced by these drugs. These results are consistent with earlier proposals that deletion of Gad2 (on 129N2 background) reduces synaptic GABA but also suggest changes in extrasynaptic receptor function.

Ethanol exerts complex effects on human physiology and health. Ethanol is not only addictive, but it is also a fetal teratogen, an adult neurotoxin, and an etiologic agent in hepatic and cardiovascular disease, inflammation, bone loss, and fracture susceptibility. A large number of genes and signaling mechanisms have been implicated in ethanol's deleterious effects leading to the suggestion that ethanol is a "dirty drug." An important question is, are there cellular "master-switches" that can explain these pleiotropic effects of ethanol? MicroRNAs (miRNAs) have been recently identified as master regulators of the cellular transcriptome and proteome. miRNAs play an increasingly appreciated and crucial role in shaping the differentiation and function of tissues and organs in both health and disease. This critical review discusses new evidence showing that ethanol-sensitive miRNAs are indeed regulatory master-switches. More specifically, miRNAs control the development of tolerance, a crucial component of ethanol addiction. Other drugs of abuse also target some ethanol-sensitive miRNAs suggesting that common biochemical mechanisms underlie addiction. This review also discusses evidence that miRNAs mediate several ethanol pathologies, including disruption of neural stem cell proliferation and differentiation in the exposed fetus, gut leakiness that contributes to endotoxemia and alcoholic liver disease, and possibly also hepatocellular carcinomas and other gastrointestinal cancers. Finally, this review provides a perspective on emerging investigations into potential roles of miRNAs as mediators of ethanol's effects on inflammation and fracture healing, as well as the potential for miRNAs as diagnostic biomarkers and as targets for therapeutic interventions for alcohol-related disorders.

Urocortin 1 (Ucn 1) is an endogenous corticotropin releasing factor (CRF)-related peptide. Ucn 1 is most highly expressed in the perioculomotor urocortin containing neurons (pIIIu), previously known as the non-preganglionic Edinger-Westphal nucleus (npEW). Various studies indicate that these cells are involved in stress adaptation and the regulation of ethanol (EtOH) intake. However, the developmental trajectory of these neurons remained unexamined. Expression of the cocaine- and amphetamine-regulated transcript (CART), which co-localizes with Ucn 1 in the perioculomotor area (pIII) has been examined prenatally, but not postnatally. The goal of the current study was to characterize the ontogenetic profile of Ucn 1 and CART during postnatal development in C57BL/6J (B6) mice. B6 mice were bred, and brains were collected at postnatal days (PND) 1, 4, 8, 12, 16, 24 and 45. Brightfield immunohistochemical staining for Ucn 1 and CART showed that Ucn 1-immunoreactivity (ir) was absent at PND 1, while CART-ir was already apparent in pIIIu at birth, a finding indicating that although the pIIIu neurons have already migrated to their adult position, Ucn 1 expression is triggered in them at later postnatal stages. Ucn 1-ir gradually increased with age, approaching adult levels at PND 16. This developmental profile was confirmed by double-immunofluorescence, which showed that Ucn 1 was absent in CART-positive cells of pIII at PND 4 and that Ucn 1 and CART are strongly but not completely co-localized in pIII at PND 24. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis confirmed that Ucn 1 mRNA levels are significantly lower at PND 4 and PND 12 than in adult animals. The lack of brain Ucn 1 immunoreactivity at birth and the gradual postnatal increase in Ucn 1 in pIIIu suggests that this peptide plays a greater behavioral role in adulthood than during the early postnatal development of an organism.

The anterior cingulate cortex (ACC) has been implicated in alcohol and drug addiction. We recently identified the small G protein K-ras as an alcohol-regulated gene in the ACC by gene expression analysis. We show here that the adiponectin receptor 2 (AdipoR2) was differentially regulated by alcohol in the ACC in a K-ras-dependent manner. Additionally, withdrawal-associated increased drinking was attenuated in AdipoR2 null mice. Intracellular recordings revealed that adiponectin increased the excitability of ACC neurons and that this effect was more pronounced during alcohol withdrawal, suggesting that AdipoR2 signaling may contribute to increased ACC activity. Altogether, the data implicate K-ras-regulated pathways involving AdipoR2 in the cellular and behavioral actions of alcohol that may contribute to overactivity of the ACC during withdrawal and excessive alcohol drinking.

Cytoplasmic lipid droplets (CLDs) are cellular structures composed of a neutral lipid core surrounded by a phospholipid monolayer of amphipathic lipids and a variety of proteins. CLDs have classically been regarded as cellular energy storage structures. However, recent proteomic studies reveal that, although many of the proteins found to associate with CLDs are connected to lipid metabolism, storage, and homeostasis, there are also proteins with no obvious connection to the classical function and typically associated with other cellular compartments. Such proteins are termed refugee proteins, and their presence suggests that CLDs may serve an expanded role as a dynamic protein storage site, providing a novel mechanism for the regulation of protein function and transport.

Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides prepared from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using the SRM mass spectrometry. Our study demonstrates that elevated chromatographic temperature results in significant improvements in the magnitude of peptide recovery for both hydrophilic and hydrophobic peptides from both simple and complex peptide matrices. Importantly, the analytical benefits for quantitative measurements in whole mouse brain matrix are demonstrated, suggesting broad utility in the proteomic analyses of complex mammalian tissues. Any improvement in peptide recovery from chromatographic separations translates directly to the apparent sensitivity of downstream mass analysis in μLC-MS/MS based proteomic applications. Therefore, the incorporation of elevated chromatographic temperatures should result in significant improvements in peptide quantification as well as detection and identification.

Reward is a concept fundamental to discussions of drug abuse and addiction. The idea that altered sensitivity to either drug-reward, or to rewards in general, contributes to, or results from, drug-taking is a common theme in several theories of addiction. However, the concept of reward is problematic in that it is used to refer to apparently different behavioural phenomena, and even to diverse neurobiological processes (reward pathways). Whether these different phenomena are different behavioural expressions of a common underlying process is not established, and much research suggests that there may be only loose relationships among different aspects of reward. Measures of rewarding effects of drugs in humans often depend upon subjective reports. In animal studies, such insights are not available, and behavioural measures must be relied upon to infer rewarding effects of drugs or other events. In such animal studies, but also in many human methods established to objectify measures of reward, many other factors contribute to the behaviour being studied. For that reason, studying the biological (including genetic) bases of performance of tasks that ostensibly measure reward cannot provide unequivocal answers. The current overview outlines the strengths and weaknesses of current approaches that hinder the conciliation of cross-species studies of the genetics of reward sensitivity and the dysregulation of reward processes by drugs of abuse. Some suggestions are made as to how human and animal studies may be made to address more closely homologous behaviours, even if those processes are only partly able to isolate 'reward' from other factors contributing to behavioural output.

While benzodiazepine intoxication alone may elicit sedative and antianxiety effects, alcohol coingestion greatly amplifies this central nervous system depression. As a result, this drug combination gained notoriety for its role in cases of facilitated sexual assault and fatal overdose. We previously validated the ability of the novel antiflunitrazepam monoclonal antibody (mAb) RCA3A3 to bind flunitrazepam (FLU) in vivo and block FLU-induced impairment of locomotion and memory. A therapeutically relevant application of this high affinity mAb (K(d,app) = 200 nM), however, is to the more tenuous indication of flunitrazepam (FLU) and alcohol cointoxication. Employing a murine behavioral model, passive immunization with mAb RCA3A3 before injection of ethanol (EtOH: low-dose, 1 g/kg, or high-dose, 1.5 g/kg), FLU (0.06 mg/kg), or a cocktail of both drugs offered partial to full restoration of motor activity levels in co-drug treated and FLU-treated mouse groups (n = 12), respectively. Whereas all drug treatments left contextual learning intact, auditory cued learning was severely disrupted. Prophylactic administration of mAb RCA3A3 prevented this deficit in cued learning in FLU-treated mice but not in the FLU- and EtOH-treated mice, in which co-drug exposure exacerbated the impairment in cued fear conditioning. To substantiate this finding, a dose-response study was performed, and the changes in locomotor activity incurred by different FLU (low-dose, 0.06 mg/kg, or high-dose, 0.09 mg/kg), EtOH (1.0 g/kg, 1.5 g/kg), and mAb RCA3A3 (14.5 mg/kg, 21.8 mg/kg) dose combinations illustrated the potentiation in motor effects by concomitant exposure to FLU and EtOH. Thus, motor activity and fear conditioning results demonstrated that both the amount of FLU left unbound by antibody and the pharmacological additivity between FLU and EtOH, a GABA mimetic, were limiting factors in the therapeutic efficacy of mAb RCA3A3. In sum, our study highlights the complex nature of psychomotor impairment upon co-drug versus singular drug exposure, which may pose a unique challenge to therapeutic treatment.

A single-voxel Carr-Purcell-Meibloom-Gill sequence was developed to measure localized T2 relaxation times of 13C-labeled metabolites in vivo for the first time. Following hyperpolarized [1-13C]pyruvate injections, pyruvate and its metabolic products, alanine and lactate, were observed in the liver of five rats with hepatocellular carcinoma and five healthy control rats. The T2 relaxation times of alanine and lactate were both significantly longer in HCC tumors than in normal livers (p \textless 0.002). The HCC tumors also showed significantly higher alanine signal relative to the total 13C signal than normal livers (p \textless 0.006). The intra- and inter-subject variations of the alanine T2 relaxation time were 11% and 13%, respectively. The intra- and inter-subject variations of the lactate T2 relaxation time were 6% and 7%, respectively. The intra-subject variability of alanine to total carbon ratio was 16% and the inter-subject variability 28%. The intra-subject variability of lactate to total carbon ratio was 14% and the inter-subject variability 20%. The study results show that the signal level and relaxivity of [1-13C]alanine may be promising biomarkers for HCC tumors. Its diagnostic values in HCC staging and treatment monitoring are yet to be explored.

Although ethanol has been considered to be an anxiolytic agent, consumption of ethanol has also been shown to increase plasma adrenocorticotropin and glucocorticoids. The corticotrophin-releasing factor (CRF) receptor 1alpha (CRF-R1) is a G protein-coupled receptor that activates adenylyl cyclase (AC), leading to adrenocorticotropin (and subsequently glucocorticoid) release into the circulation. There are nine members of the membrane-bound AC family, and the type 7 AC (AC7) is most sensitive to ethanol, which enhances the responsiveness of AC7 to G protein-coupled receptor activation. We determined the time course of ethanol's effect on plasma adrenocorticotropin and corticosterone levels in male and female AC7 transgenic (Adcy7(huTG)) mice (in which AC7 is overexpressed in neural tissue) and AC7 heterozygous knockdown [Adcy7(+/-)] mice (in which AC7 is underexpressed in neural tissue), and their respective littermate controls [wild type (WT)]. CRF-R1 mRNA and mRNA and protein for different forms of ACs were measured by using gene expression arrays, quantitative reverse transcription-polymerase chain reaction, and immunoblotting in pituitaries of all animals. Our results demonstrated increased levels of AC7 in pituitary of Adcy7(huTG) mice and decreased levels in pituitary of Adcy7(+/-) mice compared with WT animals. Male and female Adcy7(huTG) mice displayed higher plasma adrenocorticotropin and corticosterone levels than WT and/or Adcy7(+/-) mice after ethanol injection. Female mice displayed higher adrenocorticotropin and corticosterone levels after ethanol injection than males, regardless of genotype. The data provide evidence for an integral role of AC7 in the increase of plasma adrenocorticotropin and corticosterone levels during alcohol intoxication.

BACKGROUND: To study withdrawal, ethanol is usually administered chronically without interruption. However, interest has recurred in models of episodic exposure. Increasing evidence suggests that chronic intermittent exposure to ethanol leads to a sensitization effect in both withdrawal severity and ethanol consumption. The goal of the present study was to examine mouse inbred strain differences in withdrawal severity following chronic intermittent exposure using the handling-induced convulsion as the behavioral endpoint. We also sought to compare the withdrawal responses of inbred strains across acute, chronic continuous, and chronic intermittent exposure regimens. METHODS: Male mice from 15 standard inbred strains were exposed to ethanol vapor for 16 hours each day for 3 days and removed to an air chamber during the intervening 8 hours. Mice in the control groups were handled the same, except that they were exposed only to air. Daily blood ethanol concentrations were averaged for each mouse to estimate total dose of ethanol experienced. RESULTS: Across strains, mice had an average daily blood ethanol concentration (BEC) of 1.45 +/- 0.02 mg/ml and we restricted the range of this value to 1.00-2.00 mg/ml. To evaluate strain differences, we divided data into two dose groups based on BEC, low dose (1.29 +/- 0.1 mg/ml) and high dose (1.71 +/- 0.02 mg/ml). After the third inhalation exposure, ethanol-exposed and air-exposed groups were tested hourly for handling-induced convulsions for 10 hour and at hour 24 and 25. Strains differed markedly in the severity of withdrawal (after subtraction of air control values) in both dose groups. CONCLUSION: The chronic intermittent exposure paradigm is sufficient to elicit differential withdrawal responses across nearly all strains. Data from the high-dose groups correlated well with withdrawal data derived from prior acute (single high dose) and chronic continuous (for 72 hours) ethanol withdrawal studies, supporting the influence of common genes on all three responses.