by the biology. Accordingly, we studied the early phase of LTP to identify interrelated networks of genes induced at perforant path synapses in the dentate gyrus in vivo [13]. This study and others consolidated the concept that a significant and co-ordinated alteration in gene expression is triggered during LTP [16,17]. However, few analyses have extended beyond this very early time frame (e.g. [18]). In the current study, we have broadened our analysis to identify gene sets differentially regulated during the late phases of LTP (5 h and 24 h post-LTP induction), elucidating their associated canonical pathways, biological functions and clustering into gene networks. The resulting holistic view of gene expression changes over 24 h has provided insight into the regulation and function of LTP-related gene expression in the hippocampus. Analysis 1. Regulation of gene expression 20 min, 5 h and 24 h following LTP induction in vivo To determine whether there are ongoing LTP-related alter- ations in gene expression and whether overlapping or distinct gene sets are regulated in the later stages of LTP, we established time- specific LTP-related gene (LRG) datasets. Our established LTP induction paradigm is well documented to produce a very persistent form of LTP at perforant path synapses in vivo (e.g. [3]). In this study, HFS resulted in a robust potentiation over 24 h of the fEPSP slope (2163%; mean 6 SEM) and the population spike (293661%) (Figure 1). LRGs were identified by isolation and purification of total RNA from individual control and stimulated hemispheres (5 h and 24 h post-LTP induction; n = 5), followed by gene expression profiling using Affymetrix Rat 230.2 arrays. After normalization across all datasets and using a moderated paired t- test (Limma), an inclusive list of differentially expressed LRGs was produced for each time point using dual selection criteria (61.15 fold change; p,0.05). This analysis identified 190 differentially expressed genes (73% increased and 27% decreased) at 5 h post- LTP induction, and 691 differentially expressed genes at 24 h post-LTP induction (31% increased and 69% decreased) (Figure 2; Table S1, S2). Overall our analysis, when compared with a re- analysis of our 20 min-LRG set [13], suggests that there is a rapid upregulation of gene expression which persists for at least 5 h, but by 24 h the proportion of upregulated genes is attenuated, and a generalised downregulation of gene expression (Figure 2). While little literature exists regarding the specific cohorts of genes regulated in the later phases of LTP [14,18,19], we explored our datasets for previously reported LTP-regulated genes. Several known LTP-regulated genes were identified within our new datasets, and a subset was confirmed using qPCR as having temporally specific gene expression (Figure 3A). We confirmed the upregulation of the immediate-early gene HOMER, observed that the metallopeptidase inhibitor TIMP1 is upregulated with a delayed time-course, and showed that the amyloid precursor protein (APP) is downregulated specifically 24 h post-LTP induction. Next, we confirmed the differential expression of several genes, which had not previously been reported to be regulated by LTP (Figure 3B). These included LINGO1, a member of the Nogo receptor signalling complex which regulates axon regeneration, the transcription factors SP1 and BHLHE22, HDAC1, a histone deacetylase, KCNE2, an ancillary protein of voltage-gated potassium channels as well as DNAJB5 (see below). To determine the overall similarity between our datasets we used Venn diagrams and investigated the intersections formed (Figure 4A; Table S3). This analysis showed that while the 20 min and 5 h datasets contain similar numbers of differentially LTP-Related Gene Networks Figure 1. Induction of robust LTP by 506400 Hz, 10 impulse trains (HFS) at perforant path dentate granule cell synapses. Data are mean 6 standard error (n = 5) for the fEPSP (A) and population spike (B). Inset waveforms are averages of 10 sweeps (5 min) for one animal, taken during baseline (1), 45–60 min (2) and 24 h (3) after HFS. Calibration bars: 5 ms, 5 mV. doi:10.1371/journal.pone.0040538.g001 Figure 2. LTP-related gene expression 20 min, 5 h and 24 h post-LTP induction. Alterations in mRNA transcript number identified 20 min, 5 h and 24 h post-LTP, as detected using Affymetrix RAT 230 2.0 microarrays; note increase in proportion of downregulated genes at 24 h (dual selection criteria: fold change $1.15 or #0.85; p,0.05). doi:10.1371/journal.pone.0040538.g002 expressed genes, few are in common and at 24 h there are 3 fold more downregulated genes than at the earlier time points. Analysis of the temporal expression profiles of the genes common PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40538