Pattern separation is a process
Pattern separation is a process that takes place in the hippocampus and more specifically in the dentate gyrus (DG) and Cornu Ammonis region 3 (CA3) (Morris, Churchwell, Kesner, & Gilbert, 2012). The main source of input in the hippocampus is derived from the enthorinal cortex (EC) that mainly projects to the granule cystic fibrosis chloride channel in the DG. From the DG the information is sent to the CA3, and from CA3 to Cornu Ammonis region 1 (CA1) (Myers & Scharfman, 2011). The process of forming distinct representations out of overlapping stimuli can only be accomplished because the DG granule cells have small place fields and can therefore disperse the input from the EC. Subsequently, the information is relayed to the CA3 region via the mossy fiber synapses (Kheirbek et al., 2012). Animal studies showed that pattern separation is based on two types of neuronal processing between the DG and CA3 (Leutgeb, 2008). The encoding of small differences at a given location takes place at the granule cells of DG. The CA3 region adds another level of pattern separation when the differences in a location are more pronounced, by activating different neuronal subpopulations (Leutgeb, Leutgeb, Moser, & Moser, 2007).
Learning and memory involving pattern separation requires changes in synaptic plasticity and associated neuronal gene expression (Feng et al., 2010), the latter of which has been shown to depend on epigenetic alterations (Feng, Fouse, & Fan, 2007). As such, the orchestrated action of DNA methylation and demethylation could define transcription of genes related to mnemonic processes. DNA methylation is controlled by DNA methyltransferases (DNMTs), which catalyze the transfer of a methyl-group at CpG sites of DNA. Accordingly, a high degree of DNA methylation, especially at the promoter region of a gene, is often associated with reduced gene expression by preventing transcription factor binding (Watt & Molloy, 1988) or by recruitment of methyl-CpG binding domain (MBD) proteins. These proteins form a complex with histone deacetylases (HDACs), promoting histone tail deacetylation, which subsequently leads to a transformation of chromatin into a condensed, repressive state (Guoping & Hutnick, 2005). However, it remains to be elucidated whether such epigenetic mechanisms may directly affect pattern separation.
In the present study, we aimed to investigate the effect of the non-specific DNMT inhibitor RG108 on pattern separation performance and therefore provide first evidence regarding the influence of an epigenetic mechanism on pattern separation memory. Next, in order to get more insight into the effect of DNMT inhibition, we analyzed the hippocampal expression of relevant target genes for plasticity and memory function after treatment with RG108. The genes of interest were histone deacetylase 2 (Hdac2), brain-derived neurotrophic factor 1, 4 and 9 (Bdnf1, 4 and 9) and glutamate ionotropic receptor AMPA type subunit 1 (Gria1). We chose to determine the expression of Hdac2 because -HDAC2 works in close concert with DNMT’s, while the remaining genes were selected due to their association with memory function. Our results indicate that administration of RG108 increases the expression of Bdnf1, while the expression levels of the other genes remained unaltered. Finally, we opted to get a first indication whether the increase in Bdnf1 expression is accompanied with differences in the methylation pattern, by analyzing the methylation levels of 14 CpG loci at its promoter region.
Materials & methods
Discussion As shown by qPCR analysis, Bdnf1 expression was up-regulated by approximately fifty percent after injection of RG108. A possible explanation is that the overall methylation in promoter of exon I is decreased by the compound, therefore facilitating its gene expression. The rodent Bdnf gene has a complex structure consisting of eight (I-VIII) 5′ non-coding exons, each driven by a specific promoter, and one 3′ coding exon (IX). Several splice variants have been described, all consisting of the 3′ coding exons and differing in the number of 5′ non-coding exons. Interestingly, multiple promoters are postulated to allow for spatio-temporal regulation of BDNF transcripts in the CNS (Pruunsild, Kazantseva, Aid, Palm, & Timmusk, 2007). Distinct sub-cellular distribution of the different splice variants and, subsequently, the BDNF protein, is achieved by restricted regulation of Bdnf mRNA trafficking (Chiaruttini, Sonego, Baj, Simonato, & Tongiorgi, 2008). For example, BDNF1 was shown to be expressed in the soma and dendrites of neuronal cells (Chiaruttini et al., 2009, Pattabiraman et al., 2005), where it contributes to the synthesis of neurotransmitters (Loudes, Petit, Kordon, & Faivre-Bauman, 1999) and the local synthesis of BDNF (Kang et al., 1996, Tongiorgi et al., 1997), respectively. Considering the important role of BDNF in neuronal functioning, we could speculate that the observed increase in Bdnf1 expression after treatment with RG108 could account for the pro-cognitive effect of the latter in short-term OPS.