Faculty Bibliography

The aim of this study was to identify neurochemical pathways and candidate genes involved in adaptation to nicotine treatment and withdrawal. Locomotor sensitization was assessed in a nicotine challenge test after exposure to intermittent nicotine treatment and withdrawal. About 24 h after the challenge test the ventral tegmentum of the mesencephaion was dissected and processed using oligonucleotide microarrays with 22,690 probe sets (Affymetrix 430A 2.0). Quasi-congenic RQI, and donor BALB/cJ mice developed significant locomotor sensitization, while sensitization was not significant in the background partner, C57BL/6By. Comparing saline treated controls of C57BL/6ByJ and BALB/cJ by a rigorous statistical microarray analysis method we identified 238 differentially expressed transcripts. Quasi-congenic strains B6.Cb4i5-alpha4/Vad and B6.Ib5i7-beta25A/Vad significantly differed from the background strain in 11 and 11 transcripts, respectively. Identification of several cis- and trans-regulated genes indicates that further work with quasi-congenic strains can quickly lead to mapping of Quantitative Trait Loci for nicotine susceptibility because donor chromosome regions have been mapped in quasi-congenic strains. Nicotine treatment significantly altered the abundance of 41, 29, 54, and 14 ventral tegmental transcripts in strains C57BL/6ByJ, BALB/cJ, B6.Cb4i5-alpha4/Vad, and B6.Ib5i7-beta25A/Vad, respectively. Although transcript sets overlapped to some extent, each strain showed a distinct profile of nicotine sensitive genes, indicating genetic effects on nicotine-induced gene expression. Nicotine-responsive genes were related to processes including regulation of signal transduction, intracellular protein transport, proteasomal ubiquitin-dependent protein catabolism, and neuropeptide signaling pathway. Our results suggest that while there are common regulatory mechanisms across inbred strains, even relatively small differences in genetic constitution can significantly affect transcriptome response to nicotine

It is believed that drug-induced behavioral sensitization is an important process in the development of substance dependence. In order to explore mechanisms of sensitization, a mouse model of nicotine-induced locomotor sensitization was established, and effects of the sensitization process on mesencepahlic gene expression were examined. A schedule, which included 3 weeks of intermittent nicotine exposure (0.5 mg/kg, s.c.) and 3 weeks of withdrawal, resulted in locomotor sensitization. Effects of sensitization on mesencephalic expression of approximately 14,000 genes were assessed using oligonucleotide microarrays. Signal intensity differences in samples obtained from repeated nicotine- and saline-exposed animals were analyzed with z-test after False Discovery Rate (FDR) multiple test correction. Genes related to GABA-A receptors and protein phosphatases were among 68 genes showing significantly different expression levels between the saline and the nicotine groups. We hypothesize that some of the gene expression changes in the mesencephalon are involved in pathways leading to nicotine-induced sensitization. Down-regulation of GABA-A receptors induced by repeated nicotine exposure may facilitate dopaminergic neuronal transmission and may contribute to increased locomotor activity

BACKGROUND: Ethanol induces apoptosis in cultured neurons. To assess the involvement of sphingolipids and neutral lipids in the apoptotic process, ethanol-induced alterations in lipid content and metabolism were examined by using primary cultured rat cerebellar granule neurons (CGNs), human neuroblastoma SK-N-SH cells, and mouse neuroblastoma Neuro2a cells. Ethanol treatment conditions that induced apoptosis in CGNs and SK-N-SH cells but not in Neuro2a cells were used for these experiments. METHODS: Cultured neurons were treated with and without 100 mM ethanol for one to three days, and the amounts of cellular sphingolipids [ceramide, glucosylceramide (GlcCer), and sphingomyelin] and neutral lipids [cholesterol, triglyceride (TG), and cholesterol ester (ChE)] were analyzed by high-performance thin-layer chromatography, using a Coomassie brilliant blue staining method. The incorporation of [C] acetate into each lipid fraction was measured in CGNs treated with and without ethanol. Also, the effect of delipidated serum, sterols, myriocin (a serine-palmitoyltransferase inhibitor), and desipramine (an acid sphingomyelinase inhibitor) on ethanol-induced lipid changes was studied by using Neuro2a cells. RESULTS: The most prominent change common to CGN, SK-N-SH, and Neuro2a cells was ethanol-induced TG accumulation. Higher incorporation of radioactivity into TG was also observed in ethanol-treated cultures when cellular lipids were metabolically labeled with [C] acetate in CGNs. In addition, ethanol elevated ceramide levels in all these neurons. However, ethanol induced decreases in GlcCer along with the reduction of cell viability in SK-N-SH cells and CGNs, whereas it increased GlcCer in Neuro2a cells that remained viable. Myriocin, which reduced ceramide levels, attenuated ethanol-induced cell death in SK-N-SH cells. Ethanol-induced accumulation of TG was sterol-independent, whereas changes in ceramide and GlcCer were affected in Neuro2a cells by the presence of sterols in the medium. Staurosporine, which induced cell death in SK-N-SH cells, increased levels of TG, ChE, and ceramides and reduced the level of GlcCer. CONCLUSIONS: The results showing that ethanol induces accumulation of TG and ceramide in cultured neurons suggest that ethanol enhances lipogenesis and/or reduces fatty acid degradation in neurons, as previously observed in other cell types. Further, ethanol-induced changes in lipid metabolism, specifically those of ceramide and GlcCer, may be related to the ethanol-induced apoptotic pathway

BACKGROUND: Although a large body of evidence suggests a role for the opioid system in alcoholism, the precise role of mu-, delta-, kappa-, and ORL1-opioid receptors and the physiological significance of their natural genetic variation have not been identified. The method of targeted gene disruption by homologous recombination has been used to knock out (KO) genes coding for opioid receptors, and study their effects on alcohol self-administration. Here we examined the effects of targeted disruption of kappa-opioid receptor (KOR) on oral alcohol self-administration and other behaviors. METHODS: Oral alcohol, saccharin and quinine self-administration was assessed in a two-bottle choice paradigm using escalating concentrations of alcohol, or tastant solutions. In preference tests 12% alcohol, 0.033% and 0.066% saccharin, and 0.03 mM and 0.1 mM quinine solutions were used. Open-field activity was determined in an arena equipped with a computer-controlled activity-detection system. Subjects were tested for three consecutive days. Locomotor activity was assessed on days 1 and 2 (after saline injection, i.p.) and on day 3 (after alcohol injection, i.p.). Alcohol-induced locomotor activity was determined as the difference in activity between day 3 and day 2. RESULTS: Male KOR KO mice in preference tests with 12% alcohol consumed about half as much alcohol as wild-type (WT) or heterozygous (HET) mice, showed lower preference for saccharin (0.033% and 0.066%) and higher preference to quinine (0.1 mM) than WT mice. Female KOR KO mice showed similar reduction in alcohol consumption in comparison to WT and HET mice. Partial deletion of KOR in HET mice did not change alcohol consumption in comparison to WT mice. In all genotype-groups females drank significantly more alcohol than males. MANOVA of locomotor activity among KO, WT, and HET mice indicated that strain and sex effects were not significant for alcohol-induced activation (p > 0.05), while strain x sex interaction effects on alcohol-induced activation could be detected (F(1,55) = 6.07, p < 0.05). CONCLUSION: Our results indicating decreased alcohol consumption, lower saccharin preference, and higher quinine preference in KOR KO mice are in line with previous observations of opioid involvement in maintenance of food intake and raise the possibility that the deficient dynorphin/KOR system affects orosensory reward through central mechanisms which reduce alcohol intake and disrupt tastant responses, either as direct effects of absence of kappa-opioid receptors, or as effects of indirect developmental compensatory changes

BACKGROUND: Endogenous and exogenous gangliosides in the plasma affect physiologic and pathologic processes such as angiogenesis and atherogenesis. However, the genetic and environmental factors that regulate the expression of plasma gangliosides are not well known. As shown in the liver and the brain, profiles of gangliosides in the plasma may be strain-specific and can be altered by intake of alcohol. Therefore, we analyzed serum gangliosides derived from inbred mouse strains with and without alcohol treatment. METHODS: C57BL/6ByJ (B6By) and BALB/cJ mice (60-70 days old) were injected with 20% alcohol (1-6 g/kg) or saline intraperitoneally, and the ganglioside content of the serum, liver, and cerebellum was measured 4 hr after the injection. Also, the effect of oral alcohol self-administration for 18 days with escalating (3-12%) concentrations of alcohol on the serum GM1 content was studied in B6By mice. The quantification of GM1 was performed with a thin-layer chromatography-staining procedure using a cholera toxin B subunit, and the content of other gangliosides was measured after staining with resorcinol reagent. RESULTS: We found that basal GM1 (containing N-glycolylneuraminic acid) content in the serum of BALB/cJ mice (4.8 +/- 0.26 ng/microl) was 25 times higher than that of B6By mice (0.19 +/- 0.01 ng/microl); the major ganglioside in both strains was GM2. The ganglioside profile in the liver was similar to that of the serum, and the GM1 content in BALB/cJ was nine times higher than that of B6By. Both injection and oral self-administration of alcohol lowered GM1 levels in the serum. CONCLUSIONS: Endogenous ganglioside profiles in the serum are under genetic control among inbred mouse strains, and they can be altered by acute and chronic alcohol administration. These genetic and alcohol-induced differences in the plasma gangliosides, which appear to reflect ganglioside metabolism in the liver, may affect alcohol-related behaviors and pathologic processes

Results of recent studies support the notion that substance self-administration is partially a genetically controlled component of addiction tied to habit formation and cellular modification of the striatum. Aiming to define pathways among genomic, neural, and behavioral determinants of addiction, we investigated global striatal gene expression in a paradigm of oral self-administration of alcohol by using genomically very similar alcohol-nonpreferring B6.Cb(5)i(7)-alpha 3/Vad (C5A3) and alcohol-preferring B6.Ib(5)i(7)-beta 25A/Vad (I5B25A) quasi-congenic mouse strains and their progenitors, C57BL/6By (B6By) and BALB/cJ. Expression of 12,488 genes and expressed sequence tags (ESTs) was studied by using 24 high-density oligonucleotide microarrays. Transcript signal intensity differences were analyzed with z test after iterative median normalization across groups and Hochberg step-down Bonferroni procedure. As expected, striatal transcriptome differences were far more extensive between the independently derived progenitor strains than between the quasi-congenic strains and their background partner, B6By. However, the genes, which were differentially expressed between the quasi-congenic strains and their background partner, were not subsets of the progenitorial differences and were not located on the chromosome segments introgressed into the quasi-congenic strains from the donor BALB/cJ strain that have been so far defined. Although 25 transcripts showed significantly different expression between the progenitor strains, only two transcripts, phosphatidylserine decarboxylase and a hypothetical 21.2-kDa protein, and one transcript, molybdenum co-factor synthesis 2, showed significantly different expression between C5A3 and I5B25A, and between B6By and I5B25A, respectively. The latter three transcripts are not located on previously identified chromosome segments introgressed from the donor BALB/cJ strain, supporting the suggestion of trans-acting regulatory variations among strains. Exposure to alcohol did not induce statistically significant striatal gene expression changes in any of the mouse strains. In conclusion, the results support the hypothesis that in functional genomic studies the chance of detecting function-relevant genes can be increased by the comparative analysis of quasi-congenic and background strains because the number of functionally irrelevant, differentially expressed genes between genomically similar strains is reduced. Lack of statistically significant alcohol-induced changes in transcript abundance indicated that oral self-administration had subtle effects on striatal gene expression and directed attention to important implications for the experimental design of future microarray gene expression studies on complex behaviors

This white paper by eighty members of the Complex Trait Consortium presents a community's view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?

The mechanisms underlying predisposition to alcohol abuse and alcoholism are poorly understood. In this study, we evaluated the role of cannabinoid (CB1) receptors in (i) voluntary alcohol consumption, and (ii) acute alcohol-induced dopamine (DA) release in the nucleus accumbens, using mice that lack the CB1 receptor gene (CB1-/-). CB1-/- mice exhibited dramatically reduced voluntary alcohol consumption, and completely lacked alcohol-induced DA release in the nucleus accumbens, as compared to wild-type mice. The gender difference, with female mice consuming significantly more alcohol than wild-type male mice, was observed in wild-type mice, whereas this gender difference was nonexistent in CB1 mutant male and female mice. There was also a significant gender difference, with the wild-type, heterozygous, and mutant females consuming significantly more liquid and food than wild-type, heterozygous and mutant males. However, the total volume of fluid consumption and food intake did not differ between wild-type, heterozygous, and mutant mice. These results strongly suggest that the CB1 receptor system plays an important role in regulating the positive reinforcing properties of alcohol

Results of recent studies have indicated an association between voluntary alcohol intake and activities of kappa-opioid receptor systems in animal models. We assessed the possibility that genetic differences observed in alcohol preference among mouse strains are related to possible polymorphisms of the kappa-opioid receptor gene (Oprk1). We compared DNA sequences of the coding region and the promoter/regulatory region of Oprk1 among C57BL/6ByJ (B6, alcohol-preferring), BALB/cJ (alcohol-avoiding), CXBI (alcohol-avoiding), and six B6.C and B6.I Recombinant QTL Introgression (RQI) strains, which carry approximately 3% of the donor BALB/cJ genome in the background B6 genome and showed various alcohol preferences. Although there were no sequence differences in the coding region, BALB/cJ had a single nucleotide polymorphism (SNP) in the promoter region, which was not detected in other strains. The results indicate that the difference in alcohol preference between B6 and BALB/cJ is not correlated with polymorphisms of Oprk1. However, results of further studies comparing Oprk1 mRNA expression between B6 and BALB/cJ showed that Oprk1 expression is regulated differently in these strains. Also, DBA/2J mice (alcohol-avoiding) showed expression of Oprk1 mRNA subtypes (alternatively spliced) different from B6 and BALB/cJ mice. Search of the Celera Genomics database indicated that DBA/2J had several SNP sites in the promoter/regulatory regions, which might explain the different expression of Oprk1 mRNA subtypes in this strain. The strain-dependent variation in the expression of alternatively spliced genes can be a significant source of phenotypic variation of complex traits such as alcohol preference