Candidate genes, pathways and mechanisms for alcoholism: an expanded convergent functional genomics approach.

We describe a comprehensive translational approach for identifying candidate genes for alcoholism. The approach relies on the cross-matching of animal model brain gene expression data with human genetic linkage data, as well as human tissue data and biological roles data, an approach termed convergent functional genomics. An analysis of three animal model paradigms, based on inbred alcohol-preferring (iP) and alcohol-non-preferring (iNP) rats, and their response to treatments with alcohol, was used. A comprehensive analysis of microarray gene expression data from five key brain regions (frontal cortex, amygdala, caudate-putamen, nucleus accumbens and hippocampus) was carried out. The Bayesian-like integration of multiple independent lines of evidence, each by itself lacking sufficient discriminatory power, led to the identification of high probability candidate genes, pathways and mechanisms for alcoholism. These data reveal that alcohol has pleiotropic effects on multiple systems, which may explain the diverse neuropsychiatric and medical pathology in alcoholism. Some of the pathways identified suggest avenues for pharmacotherapy of alcoholism with existing agents, such as angiotensin-converting enzyme (ACE) inhibitors. Experiments we carried out in alcohol-preferring rats with an ACE inhibitor show a marked modulation of alcohol intake. Other pathways are new potential targets for drug development. The emergent overall picture is that physical and physiological robustness may permit alcohol-preferring individuals to withstand the aversive effects of alcohol. In conjunction with a higher reactivity to its rewarding effects, they may able to ingest enough of this nonspecific drug for a strong hedonic and addictive effect to occur.

Protein expression changes in the nucleus accumbens and amygdala of inbred alcohol-preferring rats given either continuous or scheduled access to ethanol.

Chronic ethanol (EtOH) drinking produces neuronal alterations within the limbic system. To investigate changes in protein expression levels associated with EtOH drinking, inbred alcohol-preferring (iP) rats were given one of three EtOH access conditions in their home-cages: continuous ethanol (CE: 24h/day, 7days/week access to EtOH), multiple scheduled access (MSA: four 1-h sessions during the dark cycle/day, 5 days/week) to EtOH, or remained EtOH-naïve. Both MSA and CE groups consumed between 6 and 6.5g of EtOH/kg/day after the 3rd week of access. On the first day of EtOH access for the seventh week, access was terminated at the end of the fourth MSA session for MSA rats and the corresponding time point (2300h) for CE rats. Ten h later, the rats were decapitated, brains extracted, the nucleus accumbens (NAcc) and amygdala (AMYG) microdissected, and protein isolated for 2-dimensional gel electrophoretic analyses. In the NAcc, MSA altered expression levels for 12 of the 14 identified proteins, compared with controls, with six of these proteins altered by CE access, as well. In the AMYG, CE access changed expression levels for 22 of the 27 identified proteins, compared with controls, with 8 of these proteins altered by MSA, as well. The proteins could be grouped into functional categories of chaperones, cytoskeleton, intracellular communication, membrane transport, metabolism, energy production, or neurotransmission. Overall, it appears that EtOH drinking and the conditions under which EtOH is consumed, differentially affect protein expression levels between the NAcc and AMYG. This may reflect differences in neuroanatomical and/or functional characteristics associated with EtOH self-administration and possibly withdrawal, between these two brain structures.

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.

Regional central nervous system densities of delta-opioid receptors in alcohol-preferring P, alcohol-nonpreferring NP, and unselected Wistar rats.

The densities of delta-opioid receptors in the central nervous system of alcohol-naive, adult, male, alcohol-preferring P, alcohol-nonpreferring NP, and Wistar rats were examined with the use of quantitative autoradiography. Slides with coronal 20-microm sections through the regions of interest were incubated in 5 nM [3H]-[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) to label delta(1)-opioid receptor sites. Nonspecific binding was determined in the presence of 10 microM naloxone. Significant differences between the P and the NP rat lines were found in numerous cortical regions, the basolateral amygdala, and the posterior hippocampus, with 10%-20% lower [3H]-DPDPE binding found in the P line. In most regions examined, binding levels in the Wistar rats were intermediate between those of the P and the NP rats. Significantly lower [3H]-DPDPE binding levels in the P rat may indicate fewer delta(1)-opioid receptors or decreased binding affinity. The lower binding in certain limbic regions, such as the basolateral amygdala and posterior hippocampus, as well as cortical differences in the P rat may be associated with the divergent alcohol drinking behaviors found between the P and the NP lines.

Long-term effects of early cocaine exposure on the light responsiveness of the adult circadian timing system.

Early cocaine exposure is associated with a wide variety of neurobehavioral and teratogenic effects. The current study was conducted to determine the long-term effects of such exposure on the hamster circadian timing system. The circadian system drives rhythms in a tremendous diversity of physiological, behavioral, and endocrine functions. The fetal circadian pacemaker has recently been shown to express a functional D1 dopamine system that is involved in maternal-fetal entrainment. Maternally administered cocaine, acting on the fetal clock, could therefore potentially have long-lasting effects on exposed offspring. Pregnant SCN-lesioned hamsters or their pups, maintained in constant dim illumination (DD), were administered cocaine (30 mg/kg, SC, N = 10 litters) or saline vehicle (N = 5 litters) from embryonic (E) day 15 [day of mating = E0] through postnatal (P) day 5. Upon weaning (P21), cocaine- and saline-treated offspring were placed in individual running wheels for a period of 5-6 weeks. Individuals were then challenged with 1-h light pulses at three circadian times (CT7, CT14, CT18). Cocaine-treated litters had a statistically significant mean phase advance of +0.32 h at CT14 compared with the mean phase delay of 2.13 h of the saline-treated litters. No significant differences were seen at the other two circadian times, although there was heterogeneity in the responses among cocaine-treated animals. This represents the first demonstration of an effect of perinatal cocaine on the circadian timing system. Together with the recent demonstration of D1 receptors in the human SCN, these findings raise the possibility that gestational cocaine abuse by humans may also lead to later disturbances in the circadian timing system.

D1-dopamine receptor binding and tyrosine hydroxylase-immunoreactivity in the fetal and neonatal hamster suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the site of an endogenous biological clock that regulates mammalian circadian rhythms. Circadian rhythms, although endogenously driven, are synchronized or entrained to daily environmental cues. Developmentally, the SCN begins to oscillate before birth and is entrained to the maternal circadian rhythm by a mechanism that is still unclear. Recent evidence in rats and hamsters suggests that a fetal dopaminergic system and D1-dopamine receptors may be involved in the process of entraining the fetal clock. The present study using [3H]SCH 23390 autoradiography and tyrosine hydroxylase (TH) immunocytochemistry determined the developmental time courses of the appearance of D1 receptor in, and catecholaminergic input to, the hamster SCN. [3H]SCH 23390 binding to D1-dopamine receptors was first detected in the fetal SCN on embryonic day (E) 15, the day before birth in this species, and persisted through adulthood. The TH immunoreactive fibers were first observed on day E15 coursing just ventral to the fetal SCN but TH-immunoreactive cells and fibers were not seen within the SCN until postnatal day (P) 5. The presence of D1-dopamine receptor binding in the fetal hamster SCN is consistent with the role of these receptors in entrainment of the fetal circadian pacemaker to maternal cues. However, a receptor-transmitter mismatch exists between D1-dopamine receptors and TH-immunoreactive fibers in the fetal SCN suggesting that the role of dopamine in maternal-fetal entrainment may be as a paracrine or humoral signal.