and clathrin-mediated endocytosis at 24 h. This suggests that the LTP-associated gene response may contribute to an alteration in the synaptic calcium response and potentially contribute to metaplasticity mechanisms proposed to facilitate or inhibit further plasticity within neuronal networks [7]. It is of note that the CaMK1 gene family, previously linked to synaptic reorganisation was differentially regulated. The conclusion that LTP-related gene expression contributes to structural alterations at synapses is strengthened by the observation that multiple pathways identified both at 5 h and 24 h converge on this or related functions. Furthermore, as a number of the differentially expressed genes (e.g. FOXO1, NUMBL, MIR24-1) or molecules predicted by the IPA software (e.g. MIR124) have reported roles in neurogenesis these highlight a potential role of gene expression in LTP- stimulated neurogenesis. LTP persistence involves an intricate interplay of molecules and this is reflected in our results, which indicate that control of LTP- regulated gene expression occurs in multiple layers. We show evidence for ongoing positive regulation of gene expression via CREB (5 h-Network 1) and NFKB (24 h: Canonical Pathway; 24 h-Network ‘‘up only’’), the functions of which appear to be at least in part the regulation of GAP43 and neurotrophic signalling respectively. These findings suggest that these two transcription factors that are activated immediately after LTP induction continue to play significant roles throughout the period of LTP consolidation. Our network analysis has also highlighted mechanisms that account for the generalised downregulation of gene expression observed at 24 h. This response may contribute to resetting the transcriptional programme in response to LTP towards underlying homeostatic mechanisms, or it may facilitate expression of the ‘‘positive’’ LTP-related features, such as neurotrophin signalling, structural modification or neurogenesis. We identified multiple pathways that mediate downregulation of gene expression. These function by directly modulating protein levels either by ubiquiti- nation, endocytosis, inhibition of protein synthesis or microRNA- driven translation arrest. Control of gene expression was also demonstrated through temporally controlled upregulation of histone acetylases and downregulation of transcription factors such as SP1. Importantly, as modification of histone acetylation has previously been shown to occur in other models of memory [57,61], our data reinforce the importance of epigenetic mecha- nisms in synaptic plasticity. In summary, our data suggest that LTP-related gene expression contributes directly to both regula- tion of calcium dynamics as well as structural or neurogenic modifications of the network, which are likely to be involve ongoing activation of the CREB and NFKB transcription factors. Furthermore, we have identified a complex set of transcriptional regulatory mechanisms that appear to coalesce to form a transcriptional environment that facilitates the consolidation of LTP. These data should provide a solid platform for the development of biologically realistic neurogenetic computational models of memory storage processes. Materials and Methods Induction of LTP in freely moving animals Ethics Statement. The surgical protocols, previously de- scribed in detail [13] were approved by the University of Otago Animal Ethics Committee (Permit number: 115–09) and were in accordance with New Zealand animal welfare legislation. All experiments were conducted on perforant path dentate gyrus synapses in adult male Sprague-Dawley rats (400–550 g, 4– 6 months old at the time of surgery). Teflon-coated stainless steel LTP-Related Gene Networks wire stimulating and recording electrodes were implanted bilater- ally to establish perforant path-evoked field potentials, which were recorded in the dentate hilus. The methods of LTP induction are the same as those described in [3] and [13], with the exception that the animals were euthanized either 5 h or 24 h following LTP induction (n = 5 per group). In order to reduce variation all animals were tetanized unilaterally so that the unstimulated hemisphere could serve as a within-animal control. Our standard experimental protocols reliably produce an increase in the fEPSP that lasts for at least several weeks [3]. RNA purification and microarray hybridization RNA isolation and array hybridization were carried out as described previously [13]. RNA was isolated from individual snap- frozen dentate gyri using TRIzol reagent (Invitrogen) followed by purification through RNeasy Mini columns (Qiagen, USA). Samples meeting our quality control criteria (average RNA integrity number .8, [62]) were biotin-labeled and hybridized to Affymetrix RAT 230 2.0 arrays at the Centre for Proteomics and Genomics, University of Auckland. RNA isolation for microRNA analysis was performed similarly, but using Norgen Total RNA purification columns (Biotek Corporation) to retain these small RNA molecules. Microarray data analysis All array data were assessed using the Bioconductor software packages [63] as described previously [13], with an initial evaluation performed using the affyQCReport package, which confirmed that all quality control measures were within the recommended ranges [64]. The Robust Multichip Average (RMA) package (version 1.16.0) was used to normalize the data derived from control and stimulated hemispheres 20 min [13], 5 h and 24 h datasets. As outlined before [13], inclusive LTP-related gene (LRG) sets suitable for network analysis (either direct or indirect interactions) were derived using dual selection criteria: a threshold fold change cutoff (1.15) and a moderated paired t-test between matched stimulated and control samples (significance criterion of p,0.05). The t-statistic was generated using the Limma package, which utilizes a standard error moderated across all genes using a simple Bayesian model and produces p-values with greater degrees of freedom and hence greater reliability [65]. More stringent selection criteria were not used in this study to avoid the risk of Type 2 error and unnecessarily limited datasets at 5 h and 24 h for network analysis. All data comply with MIAME guidelines and is available through ArrayExpress (Accession number to be confirmed). Real-time quantitative PCR: mRNA Selected genes identified as differentially expressed by DNA microarray analysis were also analysed by quantitative PCR (qPCR). RNA was extracted as described above and reverse- transcribed to first strand cDNA using Superscript III (Invitrogen). Primers were designed using Primer 3 (http://frodo.wi.mit.edu/ primer3/) and obtained from Sigma or Integrated DNA Technologies. Primer sequences are described in Table S6. qPCR was performed using SYBR green Roche mastermix on either an ABI 7300 or a Roche Lightcycler 480. qPCR samples were normalised to the housekeeping gene HPRT using the 2-DDCT method [66]. Standard curves were established and amplification and dissociation curves were analysed. Significance was assessed using Student’s t-tests with the criterion set at p,0.05. PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e40538