The synthesis of arginine vasopressin (AVP) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus is sensitive to increased plasma osmolality and a decreased blood volume, and thus is robustly increased by both dehydration (increased plasma osmolality and decreased blood volume) and salt loading (increased plasma osmolality). Both stimuli result in functional remodelling of the SON and PVN, a process referred to as functional-related plasticity. Such plastic changes in the brain have recently been associated with altered patterns of DNA methylation at CpG (cytosine-phosphate-guanine) residues, a process considered to be important for the regulation of gene transcription. In this regard, the proximal Avp promoter contains a number of CpG sites and is recognised as one of four CpG islands for the Avp gene, suggesting that methylation may be regulating Avp transcription. In the present study, we show that, in an immortalised hypothalamic cell line 4B, the proximal Avp promoter is highly methylated, and treatment of these cells with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine to demethylate DNA dramatically increases basal and stimulated Avp biosynthesis. We report no changes in the expression of DNA methyltransferases, Dnmt1 and Dnmt3a, whereas there is decreased expression of the demethylating enzyme ten-eleven-translocation 2, Tet2, in the SON by dehydration and salt loading. We found higher methylation of the SON Avp promoter in dehydrated but not salt-loaded rats. By analysis of individual CpG sites, we observed hypomethylation, hypermethylation and no change in methylation of specific CpGs in the SON Avp promoter of the dehydrated rat. Using reporter gene assays, we show that mutation of individual CpGs can result in altered Avp promoter activity. We propose that methylation of the SON Avp promoter is necessary to co-ordinate the duel inputs of increased plasma osmolality and decreased blood volume on Avp transcription in the chronically dehydrated rat.
The phenotypic differentiation between oxytocin (OT)- and vasopressin (VP)-secreting magnocellular neurosecretory cells (MNCs) from the supraoptic nucleus is relevant to understanding how several physiological and pharmacological challenges affect their electrical activity. Although the firing patterns of OT and VP neurons, both in vivo and in vitro, may appear different from each other, much is assumed about their characteristics. These assumptions make it practically impossible to obtain a confident phenotypic differentiation based exclusively on the firing patterns. The presence of a sustained outward rectifying potassium current (SOR) and/or an inward rectifying hyperpolarization-activated current (IR), which are presumably present in OT neurons and absent in VP neurons, has been used to distinguish between the two types of MNCs in the past. In this study, we aimed to analyze the accuracy of the phenotypic discrimination of MNCs based on the presence of rectifying currents using comparisons with the molecular phenotype of the cells, as determined by single-cell RT-qPCR and immunohistochemistry. Our results demonstrated that the phenotypes classified according to the electrophysiological protocol in brain slices do not match their molecular counterparts because vasopressinergic and intermediate neurons also exhibit both outward and inward rectifying currents. In addition, we also show that MNCs can change the relative proportion of each cell phenotype when the system is challenged by chronic hypertonicity (70% water restriction for 7 days). We conclude that for in vitro preparations, the combination of mRNA detection and immunohistochemistry seems to be preferable when trying to characterize a single MNC phenotype.