Microarrays can be used to monitor the expression of thousands of genes simultaneously. This technique requires high-quality RNA which can be extracted from a variety of tissues and cells including post-mortem human brain. Given the vast amount of information obtained from microarray studies, it is critical to establish valid analysis techniques to identify differentially expressed genes. This technical report describes the basic methodology and analyses used to identify such genes in human post-mortem brain tissue.

This article summarizes the proceedings of a symposium held at the 2002 Research Society on Alcoholism Meeting in San Francisco, California. The aim of this symposium was to review research on the effects of ethanol on neural stems cells and neurogenesis. Ethanol is known to alter neurogenesis during development; however, recent studies indicate that the brain forms new neurons from stem cells throughout life. Furthermore, stem cells can be transplanted into the brain, creating exciting new possibilities to study brain function. The symposium covered these research areas. Dr. Michael W. Miller reviewed knowledge on the effects of ethanol on stem cell proliferation and differentiation during development. Dr. Wu Ma described studies in culture indicating that (1) neural stem cells express functional muscarinic acetylcholine receptors (mAchR), (2) mAchR-mediated proliferation involves Ca signaling and mitogen-activated protein kinase phosphorylation, and (3) phosphoinositol-3 kinase is a downstream effector for mAchR-mediated cell proliferation via activation of Akt. Drs. Kim Nixon and Fulton T. Crews followed with in vivo studies on ethanol's effects on adult neural stem cell proliferation and differentiation. Dr. W. Michael Zawada described studies directed at dopamine neuron cell transplants into mammalian central nervous system. These studies clearly establish that ethanol has significant effects on stem cells.

This article represents the proceedings of a symposium at the 2002 ISBRA/RSA meeting in San Francisco. The organizers were Kalervo Kiianmaa and Andrey E. Ryabinin. The chairs were Kalervo Kiianmaa and Jörgen A. Engel. The presentations were (1) The role of opioidergic and dopaminergic networks in ethanol-seeking behavior, by Kalervo Kiianmaa and Petri Hyytiä; (2) Interaction between the dopamine systems in the prefrontal cortex and nucleus accumbens during ethanol self-administration, by Herman H. Samson; (3) Neurochemical and behavioral studies on ethanol and nicotine interactions, by Jörgen A. Engel, Lennart Svensson, Bo Söderpalm, and Anna Larsson; (4) Involvement of the GABA receptor in alcohol reinforcement in sP rats, by Giancarlo Colombo and Giovanni Vacca; (5) Neuroactive steroids and ethanol reinforcement, by Deborah A. Finn, and (6) Potential contribution of the urocortin system to regulation of alcohol self-administration, by Andrey E. Ryabinin and Ryan K. Bachtell.(B)

G protein-coupled inwardly rectifying potassium channels (GIRKs) provide a common link between numerous neurotransmitter receptors and the regulation of synaptic transmission. We asked whether GIRKs specify a single behavioral action that is produced by drugs acting on the diverse receptors coupled with GIRKs. By using GIRK2-null mutant mice, we found marked reduction or complete elimination of the antinociceptive (hot plate test) effects of ethanol, oxotremorine, nicotine, baclofen, clonidine, and the cannabinoid receptor agonist WIN 55,212. However, ketamine analgesia remained intact. For most drugs, there was a sex difference in antinociceptive action, and the impact of deletion of the GIRK2 channel was less in female mice. The deletion of the GIRK2 channel blocks the opioid-dependent component of stress-induced analgesia (SIA), whereas nonopioid SIA was not changed. We propose that opioid, alpha adrenergic, muscarinic cholinergic, gamma-aminobutyric acid-B, and cannabinoid receptors are coupled with postsynaptic GIRK2 channels in vivo. Furthermore, this pathway accounts for essentially all of the antinociceptive effects in males, although females appear to recruit additional signal transduction mechanisms for some analgesic drugs.

RATIONALE: Previous studies have shown that GIRK2 channel function is enhanced by ethanol and that GIRK2 null mutant mice are less sensitive to some of ethanol's effects, including anxiolysis, habituated locomotor stimulation, and acute handling-induced convulsions than wild types. Under some conditions, GIRK2 knockout mice consume more ethanol than wild types, but it is unclear whether they do so because they are more sensitive to ethanol's rewarding effects or less sensitive to its aversive effects. OBJECTIVE: To further assess the role of GIRK2 in ethanol action, GIRK2 null mutant and wild type mice were tested in conditioning models that measure the motivational effects of ethanol. METHOD: In a conditioned taste aversion (CTA) procedure, knockout and wild type mice were given ethanol (0.0, 2.0, 2.5, or 3.5 g/kg, IP) following 1-h access to saccharin every 48 h over a 10 day period. In a conditioned place preference (CPP) procedure, knockout and wild type mice were given ethanol (2.0 or 3.0 g/kg, IP) paired with one stimulus (grid or hole floor) and saline paired with the other. After four 5-min trials with each stimulus, a 60-min choice test was done. RESULTS: The results demonstrated a genotypic difference in both paradigms. In CTA, there was no difference between genotypes at 0.0 or 3.5 g/kg ethanol, but at the 2.0 and 2.5 g/kg doses, wild types developed a stronger aversion to saccharin than knockouts. In CPP, wild types developed place preference, but knockouts did not. CONCLUSIONS: These studies show that GIRK2 deletion reduced ethanol's impact in tasks that are commonly used to index the drug's rewarding and aversive effects. These findings could reflect either a learning/memory deficit or decreased sensitivity to ethanol's motivational effects in null mutant mice. The latter interpretation is more consistent with previous data showing that knockout mice consume higher doses of ethanol than wild type mice.

Acute functional tolerance to ethanol develops during a single exposure to ethanol; it has been suggested to be a predisposing factor for the development of ethanol dependence. Genetic determinants of acute functional tolerance, as well as of ethanol dependence, have been clearly demonstrated. We describe a novel approach that uses a combination of selective breeding (to segregate genes contributing to the phenotype of interest, i.e., acute functional tolerance to the incoordinating effect of ethanol), quantitative trait locus analysis (to define chromosomal regions associated with acute functional tolerance), and DNA microarray technology (to identify differentially expressed genes in the brains of the selected lines of mice) to identify candidate genes for the complex phenotype of ethanol tolerance. The results indicate the importance of a signal transduction cascade that involves the glutamate receptor delta2 protein, the Ephrin B3 ligand, and the NMDA receptor, as well as a transcriptional regulatory protein that may be induced by activation of the NMDA receptor (zinc finger protein 179) and a protein that can modulate downstream responses to NMDA receptor activation (peroxiredoxin), in mediating acute tolerance to the incoordinating effect of ethanol.

Alcoholism is a complex genetic trait; susceptibility is influenced by multiple genes of small effect. To pursue mechanistic studies, genetic animal models have been used. These models are partial, each addressing one or more of the contributing traits rather than the disease as a whole. Animal studies have modeled alcohol's rewarding effects, the development of tolerance, the pathological consequences to brain systems, and the dependence on alcohol inferred from the presence of withdrawal symptoms when the drug is removed. The classical genetic methods of inbred strain analysis and development and studies of selectively bred lines have been employed for more than 40 years. Recently, such studies have shown that a genetic tendency to experience severe withdrawal is associated with a tendency to avoid self-administration of alcohol. Also recently, attempts to identify the specific genes conferring risk or protection from alcohol's effects have been undertaken. These studies have used mapping techniques based on gene sequence polymorphisms, studies of gene expression differences, and the use of candidate gene targeting such as creation of null mutants. Studies reviewed here have mapped quantitative trait loci (QTL) for many genes affecting alcohol sensitivity, tolerance, reward, and withdrawal severity. The furthest progress in gene mapping has been made toward one withdrawal QTL on mouse chromosome 4. Using multiple congenic strains, the gene conferring increased withdrawal severity has been isolated to a region of less than 1 centiMorgan, containing fewer than 20 genes. A strong candidate gene, coding for a multiple PS095/DLG/Z0-1 (PDZ) binding domain zinc finger protein, cannot be excluded. Although many more such genes will be identified in the near future, their contribution to the mapped phenotype will be shown to be dependent on epistatic interactions with other risk genes, as well as genes in the animal's background. Progress in gene identification will also depend crucially on the precise description of the phenotypes being mapped so that their pleiotropic range of influence on the multi-behavioral phenotypic syndrome can be determined. © 2002 Wiley-Liss, Inc.

The medium spiny neurons of the nucleus accumbens receive both an excitatory glutamatergic input from forebrain and a dopaminergic input from the ventral tegmental area. This integration point may constitute a locus whereby the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors promotes drug reinforcement. Here we investigate how dopaminergic inputs alter the ethanol sensitivity of NMDA receptors in rats and mice and report that previous dopamine receptor-1 (D1) activation, culminating in dopamine and cAMP-regulated phosphoprotein-32 kD (DARPP-32) and NMDA receptor subunit-1 (NR1)-NMDA receptor phosphorylation, strongly decreases ethanol inhibition of NMDA responses. The regulation of ethanol sensitivity of NMDA receptors by D1 receptors was absent in DARPP-32 knockout mice. We propose that DARPP-32 mediated blunting of the response to ethanol subsequent to activation of ventral tegmental area dopaminergic neurons initiates molecular alterations that influence synaptic plasticity in this circuit, thereby promoting the development of ethanol reinforcement.

Previous studies have shown that ethanol enhanced [(3)H]dopamine uptake in Xenopus oocytes expressing the dopamine transporter (DAT). This increase in DAT activity was mirrored by an increase in the number of transporters expressed at the cell surface. In the present study, ethanol potentiated the function of DAT expressed in HeLa cells but inhibited the function of the related norepinephrine transporter (NET). Chimeras generated between DAT and NET were examined for ethanol sensitivity and demonstrated that a 76-amino acid region spanning transmembrane domains (TMD) 2 and 3 was essential for ethanol potentiation of DAT function. The second intracellular loop between TMD 2 and 3 of DAT, which differs from that of NET by four amino acids, was explored for possible sites of ethanol action. Site-directed mutagenesis was used to replace each of these residues in DAT with the corresponding residue in NET, and the resulting cRNA were expressed in Xenopus oocytes. We found that mutations G130T or I137F abolished ethanol potentiation of DAT function, whereas the mutations F123Y and L138F had no significant effect. These results identify novel sites in the second intracellular loop that are important for ethanol modulation of DAT activity.

.Rationale: G-protein-coupled inwardly rectifying potassium channels (GIRKs) regulate synaptic transmission and neuronal firing rates. Co-localization of GIRK2 channels and dopamine receptors in the mesolimbic system suggests a role in regulation of motor activity. Objectives: To explore the role of GIRK channels in the regulation of motor behavior. Methods: GIRK2 null mutant mice (knockout) were used. Locomotor activity in a mildly stressful situation was conducted either in a circular open field with video tracking or in standard mouse cages equipped with infrared sensors. Drugs were injected intraperitoneally or subcutaneously. Results: GIRK2 knockout mice demonstrated a transient "hyperactive" behavioral phenotype with initially higher motor activity and slower habituation in a novel situation, increased levels of spontaneous locomotor activity during dark phase in their home cages, and impaired habituation in the open-field test. After habituation, GIRK2 knockout mice showed higher motor activity, which was inhibited by the D1 receptor antagonist SCH 23390 and was more sensitive to the activating effects of the D1 receptor partial agonist SKF 38393. In a novel environment (open-field) only the highest dose of SKF38393 used (20 mg/kg) produced significant activation, perhaps due to a ceiling effect in GIRK2 knockout mice. SCH 23390 inhibited the basal activity levels of mice of both genotypes. Conclusions: Activation of the dopamine D1 receptor in a stressful environment may be stronger in GIRK2 deficient mice, and this modified function of D1 receptors may cause the transient hyperactive behavioral phenotype of these mice.

Alcoholism is a major health problem in Western countries, yet relatively little is known about the mechanisms by which chronic alcohol abuse causes the pathologic changes associated with the disease. It is likely that chronic alcoholism affects a number of signaling cascades and transcription factors, which in turn result in distinct gene expression patterns. These patterns are difficult to detect by traditional experiments measuring a few mRNAs at a time, but are well suited to microarray analyses. We used cDNA microarrays to analyze expression of approximately 10 000 genes in the frontal and motor cortices of three groups of chronic alcoholic and matched control cases. A functional hierarchy was devised for classification of brain genes and the resulting groups were compared based on differential expression. Comparison of gene expression patterns in these brain regions revealed a selective reprogramming of gene expression in distinct functional groups. The most pronounced differences were found in myelin-related genes and genes involved in protein trafficking. Significant changes in the expression of known alcohol-responsive genes, and genes involved in calcium, cAMP, and thyroid signaling pathways were also identified. These results suggest that multiple pathways may be important for neuropathology and altered neuronal function observed in alcoholism.

In previous work, we identified genetic correlations between cAMP accumulation in the cerebellum and sensitivity to the incoordinating effects of ethanol. A genetic correlation suggests that common genes underlie the phenotypes investigated. One method for provisionally identifying genes involved in a given phenotypic measure is quantitative trait locus (QTL) analysis. Using a panel of 30 BXD recombinant inbred strains of mice and the progenitors (DBA/2J and C57BL/6J), and the dowel test for ataxia, we measured the blood ethanol concentrations at the time an animal first fell from the dowel and acute functional tolerance (AFT), and investigated cAMP signaling in the cerebellum. Cyclic AMP accumulation was measured in whole-cell preparations of cerebellar minces from individual mice under basal or stimulated conditions. We conducted a genome-wide QTL analysis of the behavioral and biochemical measures with \textgreater2000 genetic markers to identify significant associations. Western blot and comparative sequencing analysis were used to compare cAMP response element binding protein (CREB) levels and protein-coding sequence, respectively. QTL analyses correlating strain means with allelic status at genetic markers identified several significant associations (p \textless 0.01). Analysis of variance revealed an effect of strain on behavioral and biochemical measures. There was a significant genetic correlation between initial sensitivity and basal cAMP accumulation in the cerebellum. We identified 6 provisional QTLs for initial sensitivity on four chromosomes, 6 provisional QTLs for AFT on four chromosomes, and 11 provisional QTLs for cAMP signaling on nine chromosomes. Two loci were found to overlap for measures of initial sensitivity and for cAMP signaling. Given the genetic correlation between initial sensitivity and basal cAMP accumulation, we investigated candidate genes in a QTL on chromosome 1. Comparative sequence analysis was performed, and protein levels were compared between C57 and DBA mice for Creb1. No significant differences were detected in coding sequence or protein levels for CREB. These results suggest that although ethanol sensitivity and cAMP signaling are determined by multiple genes, they may share certain genetic codetermination.

The overall scheme of a microarray experiment is summarized in Figure 3. The real benefit of using microarrays in research is gaining knowledge from the abundant data provided from the series of related microarray experiments. The core laboratory has produced three cDNA arrays, a mouse 15K array, a human 13K array, and a human apoptosis array with 350 plus apoptotic elements. Recently, the 15K array is being used in building a database for gene expression in several areas of the mouse brain and for studying transgenic and knockout mice. The apoptosis array has been used by cancer researchers to further elucidate changes and interactions between genes leading to cell death and cancer. The UCHSC Gene Expression Array Core is supported by the National Institute on Aging and the National Cancer Institute (Bethesda, MD) to serve all academic users and has a special interest in providing a solid technological base for genomic researchers interested in alcohol and cancer research.

Glycine receptors (GlyRs) are pentameric ligand-gated ion channels that inhibit neurotransmission in the adult brainstem and spinal cord. GlyR function is potentiated by ethanol in vitro, and a mutant GlyR subunit α1(S267Q) is insensitive to the potentiating effects of ethanol. To test the importance of GlyR for the actions of ethanol in vivo, we constructed transgenic mice with this mutation. Under the control of synapsin I regulatory sequences, transgenic expression of S267Q mutant GlyR α1 subunits in the nervous system was demonstrated using [3H]strychnine binding and immunoblotting. These mice showed decreased sensitivity to ethanol in three behavioral tests: ethanol inhibition of strychnine seizures, motor incoordination (rotarod), and loss of righting reflex. There was no change in ethanol sensitivity in tests of acute functional tolerance or body temperature, and there was no change in ethanol metabolism. Transgene effects were pharmacologically specific for ethanol, compared with pentobarbital, flurazepam, and ketamine. These results support the idea that glycine receptors contribute to some behavioral actions of ethanol and that ethanol sensitivity can be changed in vivo by transgenic expression of a single receptor subunit.

The nature of the differences between mortal somatic cells and immortal germ cell lines constitutes a major area of theoretical gerontology which has not yet received adequate attention. Weismann's theory, first stated almost exactly a century ago, was recently reconsidered by Kirkwood and Holliday. They applied modern concepts and findings on the factors regulating the accuracy of synthesis of macromolecules to explain germ line immortality. In the present paper, evidence on ageing of reproductive cells and the relationship of cytomorphogenetic events to periodic rejuvenation of germ cell lines is summarized and evaluated. Key events include the elimination or reversal of some DNA changes in germ cells through recombination and meiotic haploidization, cyclic regeneration of transcriptional and translational systems during gametogenesis and early development, and the selection of stable, viable genomes at various stages of the reproductive cycle. These rejuvenatory processes are compared and related to molecular events which differentiated somatic cells are unable to carry out.