Effect of polymorphism on expression of the neuropeptide Y gene in inbred alcohol-preferring and -nonpreferring rats

Abstract

Using animal models of alcoholism, previous studies suggest that neuropeptide Y (NPY) may be implicated in alcohol preference and consumption due to its role in the modulation of feeding and anxiety. Quantitative trait loci (QTL) analysis previously identified an interval on rat chromosome 4 that is highly associated with alcohol preference and consumption using an F2 population derived from inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats. NPY mapped to the peak of this QTL region and was prioritized as a candidate gene for alcohol-seeking behavior in the iP and iNP rats. In order to identify a potential mechanism for reduced NPY protein levels documented in the iP rat, genetic and molecular components that influence NPY expression were analyzed between iP and iNP rats. Comparing the iP rat to the iNP rat, quantitative real-time polymerase chain reaction detected significantly decreased levels of NPY mRNA expression in the iP rat in the six brain regions tested: nucleus accumbens, frontal cortex, amygdala, hippocampus, caudate-putamen, and hypothalamus. In addition, the functional significance of three previously identified polymorphisms was assessed using in vitro expression analysis. The polymorphism defined by microsatellite marker D4Mit7 in iP rats reduced luciferase reporter gene expression in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the NPY gene resulting from the D4mit7 marker polymorphism may contribute to reduced levels of NPY in discrete brain regions in the iP rats.Keywords neuropeptide Y; alcohol preference; selective breeding; mRNA expression; polymorphism; quantitative trait locus Alcoholism is a complex disorder influenced by the interaction between multiple genes and the environment, exhibiting a heritability that ranges from 50 to 60% in both men and women (Heath et al., 1997). Thus far, however, only the protective effects of the alcohol metabolizing enzymes have been consistently replicated (Foroud and Li, 1999;Thomasson et al., 1993). To identify genetic factors that influence alcoholism, the alcohol-preferring (P) and -nonpreferring (NP) lines were developed from a randomly bred closed colony of Wistar HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript rats through bidirectional selective breeding on the basis of alcohol consumption and preference (Li et al., 1991). In this model, P rats display the characteristics that are considered necessary for an animal model of alcoholism (Cicero, 1979). Subsequently, inbred P (iP) and NP (iNP) strains have been established that maintain highly discordant alcohol consumption scores.Previous studies suggest that neuropeptide Y (NPY) is implicated in the modulation of alcohol consumption in P and NP rats (Cowen et al., 2004;Thiele and Badia-Elder, 2003;Pandey et al., 2003). P rats display lower levels of NPY immunoreactivity in various regions of the brain, including the central nucleus of the amygdala, hippocampus and the frontal cortex and higher level...

Identification of Candidate Genes for Alcohol Preference by Expression Profiling of Congenic Rat Strains

“…Npy expression was lower in the congenics than in the iNP animals; it was previously shown to be lower in iP than in iNP (Spence et al, 2005). The expression of Snca was lower in the congenic animals compared with the iNP animals ( Table 2), whereas previous studies of the iP versus iNP showed higher expression in the iP animals (Liang et al, 2003), suggesting that Snca may be more prominently regulated by trans-regulatory than cis-regulatory factors.…”

“…Most of the differences were small. Neuropeptide Y (Npy) and alpha synuclein (Snca), previously identified candidate genes for alcohol consumption in the iP and iNP rats (Liang et al, 2003;Spence et al, 2005), differed in expression in this combined analysis.…”

“…However, finding the specific genes within such regions is difficult. A strategy of making congenic animal models carrying a single QTL, and then using microarray analysis to determine which genes are altered in expression, should provide a powerful approach toward gene identification (Hitzemann et al, 2004;Hoffman and Tabakoff, 2005;Spence et al, 2005). Using this combined approach dramatically decreases the number of genes that are expected to be differentially expressed because the 2 strains are identical except for the QTL sequences, thereby increasing the probability to identify differentially expressed genes contributing to a specific phenotype such as alcohol preference.…”

Genetics of Alcohol Dependence: A Review of Clinical Studies

“…NPY modulates alcohol dependence in a rat model [123,124,125], and NPY knockout mice show increased preference for alcohol [126]. In humans, some but not all genetic association studies have found evidence for an association between polymorphisms in the NPY gene on chromosome 7 and the risk for AUDs [127,128,129,130].…”

Association between Neuropeptide Y Gene Polymorphisms and Alcohol Dependence: A Case-Control Study in Two Independent Populations

“…NPY knock-out mice consumed significantly higher amounts of alcohol than wild-type mice, whereas transgenic mice that overexpress NPY showed a reduced consumption compared to wild-type mice [16]. Analysis of six brain regions of alcohol-preferring and alcohol-nonpreferring rats revealed a significant reduction in NPY mRNA expression [21]. Very few studies have tried to elucidate the role of NPY in the regulation of alcohol consumption in humans.…”