Improved prevention and treatment of drug addiction will require deeper understanding of genetic factors contributing to susceptibility to excessive drug use. Intravenous operant self-administration methods have greatly advanced understanding of behavioral traits related to addiction. However, these methods are not suitable for large-scale genetic experiments in mice. Selective breeding of mice can aggregate 'addiction alleles' in a model that has the potential to identify coordinated effects of multiple genes. We produced mouse lines that orally self-administer high (MAHDR) or low (MALDR) amounts of methamphetamine, representing the first demonstration of selective breeding for self-administration of any psychostimulant drug. Conditioned place preference and taste aversion results indicate that MAHDR mice are relatively more sensitive to the rewarding effects and less sensitive to the aversive effects of methamphetamine, compared to MALDR mice. These results validate the oral route of self-administration for investigation of the motivational effects of methamphetamine and provide a viable alternative to intravenous self-administration procedures. Gene expression results for a subset of genes relevant to addiction-related processes suggest differential regulation by methamphetamine of apoptosis and immune pathways in the nucleus accumbens of MAHDR and MALDR mice. In each line, methamphetamine reduced an allostatic state by bringing gene expression back toward 'normal' levels. Genes differentially expressed in the drug-naïve state, including Slc6a4 (serotonin transporter), Htr3a (serotonin receptor 3A), Rela [nuclear factor κB (NFκB)] and Fos (cFos), represent candidates whose expression levels may predict methamphetamine consumption and susceptibility to methamphetamine reward and aversion.

While women are more vulnerable than men to many of the medical consequences of alcohol abuse, the role of sex in the response to ethanol is controversial. Neuroadaptive responses that result in the hyperexcitability associated with withdrawal from chronic ethanol likely reflect gene expression changes. We have examined both genders for the effects of withdrawal on brain gene expression using mice with divergent withdrawal severity that have been selectively bred from a genetically heterogeneous population. A total of 295 genes were identified as ethanol regulated from each gender of each selected line by microarray analyses. Hierarchical cluster analysis of the arrays revealed that the transcriptional response correlated with sex rather than with the selected withdrawal phenotype. Consistent with this, gene ontology category over-representation analysis identified cell death and DNA/RNA binding as targeted classes of genes in females, while in males, protein degradation, and calcium ion binding pathways were more altered by alcohol. Examination of ethanolregulated genes and these distinct signaling pathways suggested enhanced neurotoxicity in females. Histopathological analysis of brain damage following ethanol withdrawal confirmed elevated cell death in female but not male mice. The sexually dimorphic response was observed irrespective of withdrawal phenotype. Combined, these results indicate a fundamentally distinct neuroadaptive response in females compared to males during chronic ethanol withdrawal and are consistent with observations that female alcoholics may be more vulnerable than males to ethanol-induced brain damage associated with alcohol abuse.

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter

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A mutant strain of Escherichia coli was created by inserting a cassette encoding sucrose sensitivity and neomycin resistance (sacB-neo) into the small-subunit rRNA-encoding gene rrs in the rrnB operon. During growth in a complex medium, the cassette was lost from the population, and a complete rrs gene was restored at a rate of 5 ؋ 10 ؊9 per cell division. Repair of this lesion required flanking regions of DNA that were similar to the six remaining intact rRNA operons and reestablished the full complement of seven rRNA operons. The relative fitness of strains with restored rrnB operons was 1 to 2% higher than that of the mutant strain. The rrnB operon normally contains a spacer region between the 16S and 23S rRNA-encoding genes that is similar in length and tRNA gene content to the spacer in rrnC, -E, and -G. In 2 of the 14 strains in which rrnB was restored, the spacer region had the same length as the spacer region in rrnA, -D, and -H. The requirement for flanking regions of nearly identical DNA and the replication of the spacer region from other rRNA operons during the repair of rrnB suggest that the restoration was accomplished via gene conversion. The rate of gene conversion was 10-fold less than the fixation of point mutations in the same region of the chromosome but was apparently sufficient to homogenize the sequences of rRNA genes in E. coli. These findings are discussed in the context of a conceptual model describing the presence of sequence heterogeneity in coevolving rRNA genes.Unlike the majority of genes in Bacteria and Archaea that are present in single copies, there are as many as 15 copies of each of the rRNA-encoding genes (25). When multiple copies of rRNA genes are present in an organism, they are nearly (Ն99%) identical in most cases, suggesting that there must be some form of homogenization of the gene sequences to remove neutral mutations that would otherwise accumulate. Apparent exceptions to this phenomenon of concerted evolution include the rRNA genes from Thermomonospora chromogena, Thermobispora bispora, and Haloarcula marismortui, in which the 16S and 23S rRNA gene sequences differ by up to 10% (11,34,45,46). It has been suggested that lateral gene transfer is responsible for the presence of divergent rRNA genes in an organism, but co-occurrences of divergent rRNA genes in microbes appear to be exceptions to the rule.The extensive regions of sequence similarity provided by repeated rRNA genes present opportunities for recombination that can lead to inversions, duplications, deletions, and transpositions of the chromosome. Recombinational events involving rRNA operons have been observed in Escherichia coli at frequencies of 10 Ϫ3 to 10 Ϫ5 in overnight liquid cultures (3,16,(18)(19)(20)(21). This high recombination frequency suggests the potential for dynamic reorganization of the E. coli chromosome, yet the overall arrangement of rRNA operons in the chromosome is remarkably stable. Comparative genomic analysis of E. coli (strain K-12) and Salmonella enterica serovar Typhimurium (strai...

Ethanol abuse can lead to addiction, brain damage and premature death. The cycle of alcohol addiction has been described as a composite consisting of three stages: intoxication, withdrawal and craving/abstinence. There is evidence for contributions of both genotype and sex to alcoholism, but an understanding of the biological underpinnings is limited. Utilizing both sexes of genetic animal models with highly divergent alcohol withdrawal severity, Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) mice, the distinct contributions of genotype/phenotype and of sex during addiction stages on neuroadaptation were characterized. Transcriptional profiling was performed to identify expression changes as a consequence of chronic intoxication in the medial prefrontal cortex. Significant expression differences were identified on a single platform and tracked over a behaviorally-relevant time course that covered each stage of alcohol addiction; i.e., after chronic intoxication, during peak withdrawal, and after a defined period of abstinence. Females were more sensitive to ethanol with higher fold expression differences. Bioinformatics showed a strong effect of sex on the data structure of expression profiles during chronic intoxication and at peak withdrawal irrespective of genetic background. However, during abstinence, differences were observed instead between the lines/phenotypes irrespective of sex. Confirmation of identified pathways showed distinct inflammatory signaling following intoxication at peak withdrawal, with a pro-inflammatory phenotype in females but overall suppression of immune signaling in males. Combined, these results suggest that each stage of the addiction cycle is influenced differentially by sex vs. genetic background and support the development of stage- and sex-specific therapies for alcohol withdrawal and the maintenance of sobriety.

Chronic alcohol abuse is associated with brain damage in a sex-specific fashion, but the mechanisms involved are poorly described and remain controversial. Previous results have suggested that astrocyte gene expression is influenced by ethanol intoxication and during abstinence in vivo. Here, bioinformatic analysis of astrocyte-enriched ethanol-regulated genes in vivo revealed ubiquitin pathways as an ethanol target, but with sexually dimorphic cytokine signaling and changes associated with brain aging in females and not males. Consistent with this result, astrocyte activation was observed after exposure in female but not male animals, with reduced S100β levels in the anterior cingulate cortex and increased GFAP(+) cells in the hippocampus. In primary culture, the direct effects of chronic ethanol exposure followed by recovery on sex-specific astrocyte function were examined. Male astrocyte responses were consistent with astrocyte deactivation with reduced GFAP expression during ethanol exposure. In contrast, female astrocytes exhibited increased expression of Tnf, reduced expression of the neuroprotective cytokine Tgfb1, disrupted bioenergetics and reduced excitatory amino acid uptake following exposure or recovery. These results indicate widespread astrocyte dysfunction in ethanol-exposed females and suggest a mechanism that may underlie increased vulnerability to ethanol-induced neurotoxicity in females.

Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated T3 levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics.