A Genetic Animal Model of Alcoholism for Screening Medications to Treat Addiction.

The purpose of this review is to present up-to-date pharmacological, genetic, and behavioral findings from the alcohol-preferring P rat and summarize similar past work. Behaviorally, the focus will be on how the P rat meets criteria put forth for a valid animal model of alcoholism with a highlight on its use as an animal model of polysubstance abuse, including alcohol, nicotine, and psychostimulants. Pharmacologically and genetically, the focus will be on the neurotransmitter and neuropeptide systems that have received the most attention: cholinergic, dopaminergic, GABAergic, glutamatergic, serotonergic, noradrenergic, corticotrophin releasing hormone, opioid, and neuropeptide Y. Herein, we sought to place the P rat's behavioral and neurochemical phenotypes, and to some extent its genotype, in the context of the clinical literature. After reviewing the findings thus far, this chapter discusses future directions for expanding the use of this genetic animal model of alcoholism to identify molecular targets for treating drug addiction in general.

Genome-wide association data suggest ABCB1 and immune-related gene sets may be involved in adult antisocial behavior.

Adult antisocial behavior (AAB) is moderately heritable, relatively common and has adverse consequences for individuals and society. We examined the molecular genetic basis of AAB in 1379 participants from a case-control study in which the cases met criteria for alcohol dependence. We also examined whether genes of interest were expressed in human brain. AAB was measured using a count of the number of Antisocial Personality Disorder criteria endorsed under criterion A from the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). Participants were genotyped on the Illumina Human 1M BeadChip. In total, all single-nucleotide polymorphisms (SNPs) accounted for 25% of the variance in AAB, although this estimate was not significant (P=0.09). Enrichment tests indicated that more significantly associated genes were over-represented in seven gene sets, and most were immune related. Our most highly associated SNP (rs4728702, P=5.77 × 10(-7)) was located in the protein-coding adenosine triphosphate-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1). In a gene-based test, ABCB1 was genome-wide significant (q=0.03). Expression analyses indicated that ABCB1 was robustly expressed in the brain. ABCB1 has been implicated in substance use, and in post hoc tests we found that variation in ABCB1 was associated with DSM-IV alcohol and cocaine dependence criterion counts. These results suggest that ABCB1 may confer risk across externalizing behaviors, and are consistent with previous suggestions that immune pathways are associated with externalizing behaviors. The results should be tempered by the fact that we did not replicate the associations for ABCB1 or the gene sets in a less-affected independent sample.

Cortical-striatal gene expression in neonatal hippocampal lesion (NVHL)-amplified cocaine sensitization.

Cortical-striatal circuit dysfunction in mental illness may enhance addiction vulnerability. Neonatal ventral hippocampal lesions (NVHL) model this dual diagnosis causality by producing a schizophrenia syndrome with enhanced responsiveness to addictive drugs. Rat genome-wide microarrays containing >24 000 probesets were used to examine separate and co-occurring effects of NVHLs and cocaine sensitization (15 mg/kg/day × 5 days) on gene expression within medial prefrontal cortex (MPFC), nucleus accumbens (NAC), and caudate-putamen (CAPU). Two weeks after NVHLs robustly amplified cocaine behavioral sensitization, brains were harvested for genes of interest defined as those altered at P < 0.001 by NVHL or cocaine effects or interactions. Among 135 genes so impacted, NVHLs altered twofold more than cocaine, with half of all changes in the NAC. Although no genes were changed in the same direction by both NVHL and cocaine history, the anatomy and directionality of significant changes suggested synergy on the neural circuit level generative of compounded behavioral phenotypes: NVHL predominantly downregulated expression in MPFC and NAC while NVHL and cocaine history mostly upregulated CAPU expression. From 75 named genes altered by NVHL or cocaine, 27 had expression levels that correlated significantly with degree of behavioral sensitization, including 11 downregulated by NVHL in MPFC/NAC, and 10 upregulated by NVHL or cocaine in CAPU. These findings suggest that structural and functional impoverishment of prefrontal-cortical-accumbens circuits in mental illness is associated with abnormal striatal plasticity compounding with that in addictive disease. Polygenetic interactions impacting neuronal signaling and morphology within these networks likely contribute to addiction vulnerability in mental illness.

A genome-wide association study of alcohol-dependence symptom counts in extended pedigrees identifies C15orf53.

Several studies have identified genes associated with alcohol-use disorders (AUDs), but the variation in each of these genes explains only a small portion of the genetic vulnerability. The goal of the present study was to perform a genome-wide association study (GWAS) in extended families from the Collaborative Study on the Genetics of Alcoholism to identify novel genes affecting risk for alcohol dependence (AD). To maximize the power of the extended family design, we used a quantitative endophenotype, measured in all individuals: number of alcohol-dependence symptoms endorsed (symptom count (SC)). Secondary analyses were performed to determine if the single nucleotide polymorphisms (SNPs) associated with SC were also associated with the dichotomous phenotype, DSM-IV AD. This family-based GWAS identified SNPs in C15orf53 that are strongly associated with DSM-IV alcohol-dependence symptom counts (P=4.5 × 10(-8), inflation-corrected P=9.4 × 10(-7)). Results with DSM-IV AD in the regions of interest support our findings with SC, although the associations were less significant. Attempted replications of the most promising association results were conducted in two independent samples: nonoverlapping subjects from the Study of Addiction: Genes and Environment (SAGE) and the Australian Twin Family Study of AUDs (OZALC). Nominal association of C15orf53 with SC was observed in SAGE. The variant that showed strongest association with SC, rs12912251 and its highly correlated variants (D'=1, r(2) 0.95), have previously been associated with risk for bipolar disorder.

Gene expression in the hippocampus of inbred alcohol-preferring and -nonpreferring rats.

The hippocampus is sensitive to the effects of ethanol and appears to have a role in the development of alcohol tolerance. The objective of this study was to test the hypothesis that there are innate differences in gene expression in the hippocampus of inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats that may contribute to differences in sensitivity to ethanol and/or in the development of tolerance. Affymetrix microarrays were used to measure gene expression in the hippocampus of alcohol-naive male iP and iNP rats in two experiments (n=4 and 6 per strain in the two experiments). Combining data from the two experiments, there were 137 probesets representing 129 genes that significantly differed (P < or = 0.01); 62 probesets differed at P < or = 0.001. Among the 36% of the genes that were expressed more in the iP than iNP rat at this level of significance, many were involved in cell growth and adhesion, cellular stress reduction and anti-oxidation, protein trafficking, regulation of gene expression, synaptic function and metabolism. Among the 64% of the genes that had lower expression in the hippocampus of iP than iNP rats were genes involved in metabolic pathways, cellular signaling systems, protein trafficking, cell death and neurotransmission. Overall, the data indicate that there are significant innate differences in gene expression in the hippocampus between iP and iNP rats, some of which might contribute to the differences observed in the development of alcohol tolerance between the selectively bred P and NP lines.

Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening systemic illness of abrupt onset and unknown cause. Proteolysis of the blood-clotting protein von Willebrand factor (VWF) observed in normal plasma is decreased in TTP patients. However, the identity of the responsible protease and its role in the pathophysiology of TTP remain unknown. We performed genome-wide linkage analysis in four pedigrees of humans with congenital TTP and mapped the responsible genetic locus to chromosome 9q34. A predicted gene in the identified interval corresponds to a segment of a much larger transcript, identifying a new member of the ADAMTS family of zinc metalloproteinase genes (ADAMTS13). Analysis of patients' genomic DNA identified 12 mutations in the ADAMTS13 gene, accounting for 14 of the 15 disease alleles studied. We show that deficiency of ADAMTS13 is the molecular mechanism responsible for TTP, and suggest that physiologic proteolysis of VWF and/or other ADAMTS13 substrates is required for normal vascular homeostasis.

DNA microarray analysis of differential gene expression of 6-year-old rat neural striatal progenitor cells during early differentiation.

EGF-responsive striatal progenitor cells from rat brain have been maintained in culture in the form of neurospheres for six years without exhausting their renewal capacity. The events surrounding differentiation of stem cells in the brain after a long progenitorship remain a mystery. Using DNA microarray analysis we investigated differential gene expression, comparing progenitor cells in their neurosphere state with the cells 24 hours after induction of differentiation. Eighty-one genes showed increased expression in the differentiated condition. Genes associated with cellular growth, neurite outgrowth, and synaptogenesis were activated, including both anti-apoptotic and pro-apoptotic genes. Two transmitter- related genes, acetylcholine receptor-beta and glutamate receptor-beta-unit were also elevated-, these genes not only fit the profile of early neural development, but also reflect the characteristics of striatal neurons. In addition, there are approximately 30 expressed sequence tags (ES7) increased during neural differentiation. Forty-seven genes showed decreased expression; half of them are known genes related to the cell cycle, cell adhesion, transcription, and signaling. Tbe signaling and cell cycle genes may be responsible for the life-long self-renewal. These data demonstrate for the first time that life-long quiescent stem cells retain the potential to become activated and develop into specific types of brain cells. The six-year long-term neural stem cells are an excellent model for studying developmental neurobiological processes and aging.

Parametric linkage analysis and disequilibrium methods to identify loci for complex disease.

A two-step process was used to find loci contributing to the qualitative disease phenotype in the Genetic Analysis Workshop (GAW) 12 simulated data. The first step used parametric linkage analysis with a limited number of dominant and recessive models to detect linkage to chromosomal regions. Subsequently, a subset of the simulated biallelic sequence polymorphisms was used for transmission/disequilibrium tests and to build haplotypes to fine map the disease-predisposing polymorphism(s). A haplotype, strongly associated with the disease phenotype whose proximal end was within 39 base pairs of the functional allele for simulated major gene 6, was identified in the isolated population.

Chronic life stress alters sympathetic, neuroendocrine, and immune responsivity to an acute psychological stressor in humans.

OBJECTIVE: Life stress is hypothesized to alter the dynamic regulation of the autonomic, neuroendocrine, and immune systems. This study examined the effects of antecedent chronic life stress on psychological and physiological responsivity after acute challenge with a psychological stressor. METHOD: Using a within-subject mixed design, male volunteers with (N = 12) and without chronic life stress (N = 11) were administered a 12-minute laboratory stressor (mental arithmetic) vs a video control. RESULTS: Acute psychological stress induced subjective distress, increases of circulating concentrations of epinephrine, norepinephrine, beta-endorphin, adrenocorticotropic hormone (ACTH), and cortisol, and a selective redistribution of natural killer (NK) cells into the peripheral blood as compared with the video control condition. Although the two groups were almost identical at baseline in psychological, sympathetic, neuroendocrine, and immune domains, the chronic stress group showed greater subjective distress, higher peak levels of epinephrine, lower peak levels of beta-endorphin and of NK cell lysis, and a more pronounced redistribution of NK cells in response to the acute psychological challenge than the controls. Furthermore, the acute stressor induced a protracted decline in NK lysis per NK cell in the chronic stress group but had no effect in the controls. CONCLUSIONS: In summary, when persons who are undergoing chronic life stress are confronted with an acute psychological challenge, an exaggerated psychologic and peak sympathomedullary reactivity occurs that is associated with decrements in individual NK cell function and is protracted beyond termination of the stressor and sympathomedullary recovery.

Partial night sleep deprivation reduces natural killer and cellular immune responses in humans.

Prolonged and severe sleep deprivation is associated with alterations of natural and cellular immune function. To determine whether alterations of immune function also occur after even a modest loss of sleep, the effects of early-night partial sleep deprivation on circulating numbers of white blood cells, natural killer (NK) cell number and cytotoxicity, lymphokine-activated killer (LAK) cell number and activity, and stimulated interleukin-2 (IL-2) production were studied in 42 medically and psychiatrically healthy male volunteers. After a night of sleep deprivation between 10 P.M. and 3 A.M., a reduction of natural immune responses as measured by NK cell activity, NK activity per number of NK cells, LAK activity, and LAK activity per number of LAK precursors (CD16,56, CD25) was found. In addition, concanavalin A-stimulated IL-2 production was suppressed after sleep deprivation due to changes in both adherent and nonadherent cell populations. After a night of recovery sleep, NK activity returned to baseline levels and IL-2 production remained suppressed. These data implicate sleep in the modulation of immunity and demonstrate that even a modest disturbance of sleep produces a reduction of natural immune responses and T cell cytokine production.