Nicotine-induced conditioned place preference involves dynamic changes in DNA methylation and hydroxymethylation in the zebrafish brain

Abstract

Modifications of the DNA methylation/demethylation cycle are fundamental for synaptic remodelling in memory-related processes. Cytosine methylation and demethylation marks epigenetically regulate gene expression and play key roles in addiction-like behaviours. DNA methyltransferases (DNMTs) catalyse methylation of DNA-cytosines (5mC), while ten-eleven translocation (TET) enzymes catalyse DNA demethylation to 5-hydroxymethylcytosine (5hmC) and further cytosine oxidations. 5hmC is stable and abundant in neurons and regulates gene expression. We examined whether conditioned place preference (CPP) to nicotine regulates 5mC and 5hmC levels in the zebrafish brain. Levels of TET1 mRNA were upregulated while levels of TET2, GADD45, DNMT, and MeCP2 mRNAs were downregulated in the zebrafish brain trained in the nicotine-CPP task. Levels of the α6 and α7 subunits of the nAChR and NMDAR1 mRNAs were also upregulated shortly after the nicotine-CPP test. Specific nuclei of the reward circuit, particularly the habenula (Hb) and raphe nucleus, and the tectum opticum (TeO) showed increases in the 5hmC mark in neuronal subpopulations induced by nicotine-CPP. Nicotine-CPP also increased, to a lesser extent, the number of cell nuclei containing high levels of 5mC in the Hb and TeO. Other reward structures, the posterior tubercular and interpeduncular nuclei, showed decreased numbers of cell nuclei containing high levels of 5mC with no changes in the number of cell nuclei showing high levels of 5hmC. Nicotine-CPP increased 5hmC levels and decreased 5mC levels in the whole brain of zebrafish. In this report, we demonstrate that the establishment of nicotine preference in the zebrafish brain is associated with changes in DNA methylation marks in neurons of the reward circuit, including the upregulation and downregulation of the 5mC mark, along with a net increase in the 5hmC mark. The specific changes in the methylation mark profile depend on the reward structure examined.