The experiments to be described have been concerned with the effect of vagus stimulation in the isolated heart and auricles of the rabbit. In the vagus-heart experiments the action of cocaine, of hexamethonium and of the cardiac glycoside ouabain was studied.As recently as 1953, Obrink & Essex state that 'it has become a widely accepted fact that stimulation of the vagi does not influence an isolated heart after 20-30 min. The reason for the transient response is not known.' Not many attempts to study this question have been found in the literature. Effects of vagal stimulation on perfused isolated heart preparations have been described by a number of workers (Cullis & Tribe, 1913;Middleton, 1947;Perry and Talesnik, 1953;Perry & Reinert, 1954), but of these only Middleton made a statement concerning the length of time his preparations worked satisfactorily. He perfused the cat's heart and found that vagal effects were obtained for 6 hr provided the heart remained in situ, but when the heart was isolated, vagal effects were not obtained after 10-30 min. Using the method now to be described it was possible to make observations for periods up to 9 hr. METHODS Rabbits were anaesthetized with urethane. The vagi were cut in the neck and dissected free from the surrounding tissue to the level of the aortic arch. The thoracic contents were excised and the cardiac ends of the vagi and the heart were cleared of as much tissue as possible, but leaving the root of the lungs and a short piece of trachea still adhering to the auricles. In this way the finer cardiac branches of the vagi were undisturbed. Finally, the aorta was cannulated, and the heart perfused by the usual Langendorff method. The preparation behaved satisfactorily provided that the final cleaning took no longer than 20 min.To prevent drugs from affecting the nerves, the perfusion fluid for the heart and the fluid bathing the stimulating electrodes were supplied by separate systems. These both included large chambers for saturating the solutions with the gas mixture. Having been warmed to a temperature of 350 C, fluid reached the heart with a pressure of 40 cm of water. The fluid supplied to the electrodes was heated by part of the water jacket which surrounded the heart. The flow of fluid

Despite extensive progress in Huntington's disease (HD) research, very little is known about the association of epigenetic variation and HD pathogenesis in human brain tissues. Moreover, its contribution to the tissue-specific transcriptional regulation of the huntingtin gene (HTT), in which HTT expression levels are highest in brain and testes, is currently unknown. To investigate the role of DNA methylation in HD pathogenesis and tissue-specific expression of HTT, we utilized the Illumina HumanMethylation450K BeadChip array to measure DNA methylation in a cohort of age-matched HD and control human cortex and liver tissues. In cortex samples, we found minimal evidence of HD-associated DNA methylation at probed sites after correction for cell heterogeneity but did observe an association with the age of disease onset. In contrast, comparison of matched cortex and liver samples revealed tissue-specific DNA methylation of the HTT gene region at 38 sites (FDR < 0.05). Importantly, we identified a novel differentially methylated binding site in the HTT proximal promoter for the transcription factor CTCF. This CTCF site displayed increased occupancy in cortex, where HTT expression is higher, compared with the liver. Additionally, CTCF silencing reduced the activity of an HTT promoter-reporter construct, suggesting that CTCF plays a role in regulating HTT promoter function. Overall, although we were unable to detect HD-associated DNA methylation alterations at queried sites, we found that DNA methylation may be correlated to the age of disease onset in cortex tissues. Moreover, our data suggest that DNA methylation may, in part, contribute to tissue-specific HTT transcription through differential CTCF occupancy.

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