Functional gene expression differences between inbred alcohol-preferring and –non-preferring rats in five brain regions

Abstract

The objective of this study was to determine if there are innate differences in gene expression in selected CNS regions between inbred alcohol-preferring (iP) and –non-preferring (iNP) rats. Gene expression was determined in the nucleus accumbens (ACB), amygdala (AMYG), frontal cortex (FC), caudate-putamen (CPU), and hippocampus (HIPP) of alcohol-naïve adult male iP and iNP rats, using Affymetrix Rat Genome U34A microarrays (n = 6/strain). Using Linear Modeling for Microarray Analysis with a false discovery rate threshold of 0.1, there were 16 genes with differential expression in the ACB, 54 in the AMYG, 8 in the FC, 24 in the CPU, and 21 in the HIPP. When examining the main effect of strain across regions, 296 genes were differentially expressed. Although the relatively small number of genes found significant within individual regions precluded a powerful analysis for over-represented Gene Ontology categories, the much larger list resulting from the main effect of strain analysis produced 17 over-represented categories (P < .05), including axon guidance, gliogenesis, negative regulation of programmed cell death, regulation of programmed cell death, regulation of synapse structure function, and transmission of nerve impulse. Co-citation analysis and graphing of significant genes revealed a network involved in the neuropeptide Y (NPY) transmitter system. Correlation of all significant genes with those located within previously established rat alcohol QTLs revealed that of the total of 313 significant genes, 71 are located within such QTLs. The many regional and overall gene expression differences between the iP and iNP rat lines may contribute to the divergent alcohol drinking phenotypes of these rats.

Introduction

Alcoholism and alcohol abuse are complex disorders that result from a combination of genetic and environmental factors. Selective breeding strategies for ethanol preference have yielded divergent rat lines that possess different frequencies of genes that impact ethanol preference, whereas the frequency of trait-irrelevant genes remains randomly distributed (Lumeng et al., 1977). The alcohol-preferring (P) and alcohol–non-preferring (NP) rat lines were established from a randomly bred, closed colony of Wistar rats using free-choice access to 10% (vol/vol) ethanol and water (Lumeng et al., 1977). P rats meet the proposed criteria (Cicero, 1979) for an animal model of alcoholism (reviewed in McBride and Li, 1998, Murphy et al., 2002). In brief, the P line of rats (1) consumes in excess of 5 g ethanol/kg body weight/day, attaining blood alcohol concentrations in the range of 50–200 mg%; (2) works to obtain ethanol when food and water are freely available; (3) consumes ethanol for its pharmacological effects, and not solely for caloric value nor taste or odor properties; (4) develops functional and metabolic tolerance; (5) develops physical dependence; and (6) demonstrates robust relapse ethanol drinking after a period of abstinence. On the other hand, NP rats consume less than 1 g ethanol/kg/day and do not attain measurable blood alcohol concentrations under free-choice conditions. Compared to NP rats, P rats are more sensitive to the low-dose stimulating effects of ethanol (Rodd et al., 2004, Waller et al., 1986), less sensitive to the high-dose motor impairing effects of ethanol (Lumeng et al., 1982), and develop acute tolerance more rapidly (Waller et al., 1983).

Innate differences in neurotransmitter and receptor systems in several brain regions have been reported between the selectively bred P and NP rat lines (reviewed in McBride and Li, 1998, Murphy et al., 2002). P rats have reduced serotonin (5-HT) and dopamine (DA) innervations (Zhou et al., 1991, Zhou et al., 1994a, Zhou et al., 1994b, Zhou et al., 1995), as well as differences in 5-HT (McBride et al., 1993a, McBride et al., 1994, McBride et al., 1997, Wong et al., 1993), DA (McBride et al., 1993b), and opioid (McBride et al., 1998, Strother et al., 2001) receptors. Furthermore, neuropeptide Y (NPY) (Ehlers et al., 1998), corticotropin-releasing factor (Ehlers et al., 1992), neurotensin (Ehlers et al., 1999), substance P, and neurokinin levels (Slawecki et al., 2001) are all significantly lower in CNS regions of P compared to NP rats. Additionally, higher functional neuronal activity has been found in numerous brain regions of the P rat compared to the NP rat (Smith et al., 2001, Strother et al., 2005).

Witzmann et al. (2003) examined differences in protein levels in the hippocampus (HIPP) and nucleus accumbens (ACB) of alcohol-naïve inbred-P (iP) and inbred-NP (iNP) rats, and found that almost all of the proteins that differed were lower in the iP rats compared to the iNP rats. Those proteins that could be identified were involved in many key aspects of neuronal function such as metabolism, cell signaling, and protein transport, which may suggest that there are basic differences in synaptic transmission mechanisms between the two rat strains (Witzmann et al., 2003). Edenberg et al. (2005) compared gene expression differences in the HIPP of two different strains of iP and iNP rats, when microarray analyses were conducted several months apart. The results indicated excellent repeatability of the assay. Genes involved in cell growth and adhesion, cellular stress reduction and antioxidation, protein trafficking, cellular signaling pathways, and synaptic function were differentially expressed in the HIPP (Edenberg et al., 2005). Worst et al. (2005) reported on the transcriptome analysis in the anterior cerebral cortex of alcohol-naïve Alko, alcohol (AA) and Alko, nonalcohol (ANA) rats, and found differences in mRNA levels between the AA and ANA rats that could alter transmitter release (e.g., vesicle-associated membrane protein 2, syntaxin 1, syntaxin binding protein). Kerns et al. (2005) examined gene expression differences in response to acute ethanol in the ACB, prefrontal cortex, and ventral tegmental area of C57BL/6J and DBA/2J mice, which have high and low alcohol drinking characteristics, respectively. Ethanol-regulated genes were region specific and involved in glucocorticoid signaling, neurogenesis, myelination, neuropeptide signaling, and retinoic acid signaling. Gene expression profiles were also reported for whole brain of inbred long-sleep and inbred short-sleep mice (Xu et al., 2001). A total of 41 genes or expressed sequence tags (ESTs) displayed significant differences between these inbred strains of mice. Expression of genes encoding tyrosine protein kinase and ubiquitin carboxyl terminal hydrolase was higher in the brain of inbred long-sleep compared to short-sleep mice. In a comprehensive transcriptome meta-analysis of different mice strains, Mulligan et al. (2006) identified several cis-regulated candidate genes for an alcohol preference quantitative trait loci (QTL) on chromosome 9.

Portions of the present data have been used for comparison analyses in two recently published studies, although none of the present data have been presented in duplicate form. In one study, the present HIPP data were used with other HIPP data to evaluate the reliability of the microarray analysis, when assays were conducted months apart (Edenberg et al., 2005). In the second study (Rodd et al., 2006), the data were used as part of a convergent functional genomics approach to identify common genes across different experimental approaches and between human and animal findings. In that study, however, the iP–iNP data were not analyzed using rigorous statistical criteria (for a gene to be considered significant, an false discovery rate (FDR)-uncorrected P < .05 was considered sufficient), and the results were presented only in a summarized format, which were then integrated with information from other studies. As the P and NP lines are well established animal models in the alcohol field, we believe it is important that the present findings, derived using rigorous region-by-region analyses, are presented because they yield a much more complete and statistically reliable picture of the genetic factors involved in the high and low alcohol drinking behavior in these rat lines.

The objective of the present study was to determine if there are innate differences between inbred P and NP rats in the expression of functionally relevant genes in selected brain regions. The current study focuses on five distinct brain regions: the ACB, caudate-putamen (CPU), amygdala (AMYG), HIPP, and frontal cortex (FC). These regions were selected based on their inclusion in the mesolimbic and mesocortical systems, both of which are critically important in the initiation and maintenance of goal directed and reward mediated behaviors (reviewed in Bonci et al., 2003; and Maldonado, 2003).