Glyoxalase 1 and glutathione reductase 1 regulate anxiety in mice.

Anxiety and fear are normal emotional responses to threatening situations. In human anxiety disorders--such as panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, social phobia, specific phobias and generalized anxiety disorder--these responses are exaggerated. The molecular mechanisms involved in the regulation of normal and pathological anxiety are mostly unknown. However, the availability of different inbred strains of mice offers an excellent model system in which to study the genetics of certain behavioural phenotypes. Here we report, using a combination of behavioural analysis of six inbred mouse strains with quantitative gene expression profiling of several brain regions, the identification of 17 genes with expression patterns that correlate with anxiety-like behavioural phenotypes. To determine if two of the genes, glyoxalase 1 and glutathione reductase 1, have a causal role in the genesis of anxiety, we performed genetic manipulation using lentivirus-mediated gene transfer. Local overexpression of these genes in the mouse brain resulted in increased anxiety-like behaviour, while local inhibition of glyoxalase 1 expression by RNA interference decreased the anxiety-like behaviour. Both of these genes are involved in oxidative stress metabolism, linking this pathway with anxiety-related behaviour.

Brain-specific knock-out of hypoxia-inducible factor-1alpha reduces rather than increases hypoxic-ischemic damage.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays an essential role in cellular and systemic O(2) homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1alpha in the brain, we exposed adult mice with late-stage brain deletion of HIF-1alpha to hypoxic injuries. Contrary to expectations, the brains from the HIF-1alpha-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1alpha can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1alpha-deficient mice. Although the role of HIF-1alpha has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1alpha in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1alpha-deficient mice is mechanistically consistent with a predominant role of HIF-1alpha as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1alpha, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR.

In situ hybridization (ISH) is an essential technique for mapping gene expression in the brain. Although many ISH protocols provide for quantitative analysis of individual mRNAs in different brain regions or across experimental conditions, this technique has lacked the necessary standardization for quantitative comparisons between different mRNA transcripts. We have developed a standardized quantitative ISH (SQuISH) protocol that utilizes multiple radioactive oligonucleotide probes, providing for increased sensitivity, decreased background and accurate comparison of relative mRNA levels. We evaluated the SQuISH protocol against a riboprobe-based ISH procedure by comparing the mRNA expression levels in the brain for two transcripts, insulin receptor substrate p53 (IRSp53) and Calsenilin. The results of these two methods were then validated by real-time quantitative PCR. Both protocols exhibited identical mRNA expression patterns for IRSp53 and Calsenilin. In three brain regions analyzed, the levels of IRSp53 mRNA expression were approximately 1.5-fold higher with the riboprobe-based ISH than with the SQuISH procedure, although the relative abundance in regional expression levels was similar between the two methods. In contrast, the levels of Calsenilin mRNA expression were 10-17-fold higher with the riboprobe-based ISH than with the SQuISH procedure and the relative abundance in regional expression levels was different. When compared to the real-time PCR results, the SQuISH trade mark method showed almost identical relative levels of IRSp53 to Calsenilin mRNA in all three brain regions analyzed, while the riboprobe-based procedure showed a completely opposite trend. These results support the accuracy of the SQuISH protocol for determining relative mRNA levels in the brain.

Low rates of reporting commercial bias by physicians following online continuing medical education activities.

BACKGROUND: Concerns have been raised about bias in commercially supported continuing medical education (CME) activities, although the data are sparse about whether such bias exists, or if so, its extent. METHODS: Postactivity CME evaluation surveys were analyzed to quantitate reporting rates of bias, overall and by funding source. RESULTS: 5Of 1,621,647 physicians who participated in online CME activities, 1,064,642 (65.7%) completed the evaluation surveys and 5.9% reported no opinion. The affirmative rates of physician perception of bias were 0.63% overall, a weighted average of 0.84% for activities developed with and 0.48% for those developed without commercial support, a difference of 0.36% (P <.001, 95% confidence interval, 0.33-0.39). Among the subgroup who strongly disagreed that there is no bias, the difference between commercial (0.17%) and noncommercial (0.11%) funding was 0.06% (P <.001, 95% confidence interval, 0.05-0.08, P <.05), smaller than the overall difference. CONCLUSIONS: These data demonstrate that about 93% of physician participants affirmatively claim to perceive no commercial bias following online CME activities, over 99% if no opinion is included, overall and regardless of funding source.

Adult mouse brain gene expression patterns bear an embryologic imprint.

The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional "imprint" consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior-posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org).

Glutathione S-transferase 8-8 expression is lower in alcohol-preferring than in alcohol-nonpreferring rats.

OBJECTIVE: A primary focus of alcohol research is to provide novel targets for alcohol treatment by identifying genes that predispose individuals to drink alcohol. Animal models of alcoholism developed by selective breeding are invaluable tools to elucidate both the genetic nature and the underlying biological mechanisms that contribute to alcohol dependence. These selected lines (high alcohol preferring and low alcohol preferring) display phenotypic and genetic differences that can be studied to further our understanding of alcohol preference and related genetic traits. By combining molecular techniques, genetic and physiological factors that underlie the cause of alcoholism can be identified. METHODS: Total gene expression analysis was used to identify genes that are differentially expressed in specific brain regions between alcohol-naive, inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats. Quantitative reverse transcriptase-polymerase chain reaction, in situ hybridization, Western blot, and sequence analysis were used to further characterize rat glutathione S-transferase 8-8 (rGST 8-8). RESULTS: Lower expression of rGST 8-8 mRNA was observed in discrete brain regions of iP compared with iNP animals, and these expression differences were confirmed. To determine additional expression patterns of rGST 8-8, we used in situ hybridization. Rat GST 8-8 was highly expressed in hippocampus, the choroid plexus of the dorsal third ventricle and the lateral ventricle, and ependymal cells along the dorsal third ventricle and the third ventricle. Western blot analysis showed that rGST 8-8 protein levels were lower in the hippocampus and the amygdala of iP compared with iNP. A silent single-nucleotide polymorphism in the coding region and three single-nucleotide polymorphisms in the 3'-UTR were identified in the rGST 8-8 cDNA. CONCLUSION: There is regional variation of rGST 8-8 expression in the brain, at both the mRNA and protein level, and the iP strain has lower innate rGST 8-8 levels than the iNP strain in discrete brain regions.

alpha-Synuclein maps to a quantitative trait locus for alcohol preference and is differentially expressed in alcohol-preferring and -nonpreferring rats.

Total gene expression analysis (TOGA) was used to identify genes that are differentially expressed in brain regions between the alcohol-naive, inbred alcohol-preferring (iP), and -nonpreferring (iNP) rats. alpha-Synuclein, expressed at >2-fold higher levels in the hippocampus of the iP than the iNP rat, was prioritized for further study. In situ hybridization was used to determine specific brain regions and cells expressing alpha-synuclein in the iP and iNP rats. Similar to alpha-synuclein mRNA levels, protein levels in the hippocampus were higher in iP rats than iNP rats. Higher protein levels were also observed in the caudate putamen of iP rats compared with iNP rats. Sequence analysis identified two single nucleotide polymorphisms in the 3' UTR of the cDNA. The polymorphism was used to map the gene, by using recombination-based methods, to chromosome 4, within a quantitative trait locus for alcohol consumption that was identified in the iP and iNP rats. A nucleotide exchange in the iNP 3' UTR reduced expression of the luciferase reporter gene in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the alpha-synuclein gene may contribute to alcohol preference in the iP rats.