LTP-Related Gene Networks Figure 4. Temporal relationship of LTP-regulated datasets. (A) Venn Diagram showing 3 datasets largely contain temporally specific genes. (B) Heatmap displaying average gene expression values (expressed as log fold change) of overlapping genes across the timepoints. doi:10.1371/journal.pone.0040538.g004 functions across our full temporal range using the most current versions of the software. 2.1 Ingenuity Pathway Analysis: Biological Functions. Biological Function analysis (Figure 5) highlighted the contribution that regulated gene expression plays in the maintenance of LTP. The function Gene expression was well above the threshold value within all three LRG datasets. Interestingly, Cell-Cell Signalling & Interaction, Molecular Transport and Protein Synthesis were all more significantly regulated at 24 h. Examination of the genes represented within the latter set (Table S4) indicate a potential downregulation of protein synthesis mediated via regulation of the pre-initiation complex and charging of tRNA with their cognate amino acids (EIF2, EPRS, EIF3) as well as a generalized disruption of the 40S initiation complex via reduction in levels of the scaffolding factor, eIF4G. 2.2 Ingenuity Pathway Analysis: Canonical Pathway Analysis and the Database for Annotation, Visualization and Integrated Discovery. The Canonical Pathways identified within the 20 min-LRG set analysis were associated with Cell growth, Cell death, Development and PDGF signalling (Figure 6). Interestingly, interleukin 6 (IL6) signalling was identified specifi- cally within our 20 min-LRG set. This supports the observation that immune response-related genes are implicated in the LTP stabilization process [16]. Canonical analysis also highlighted an early involvement of Neurotrophin/TRK signalling in the LTP stabilization process, as previously described [23,24]. Canonical pathway analysis of the 5 h-LRG set identified two separate pathways associated with G-protein coupled signalling and calcium dynamics (cAMP, Ca2+ signalling), with the majority of the genes upregulated (Figure 6; Table S5). In support of these findings, when the 5 h-LRG set was submitted to functional analysis using DAVID software, a Calcium and Calmodulin annota- tion cluster was identified as significantly enriched (enrichment score: 1.42; see Figure S1). Furthermore, DAVID analysis identified clusters of genes with annotations related to Ion channels (enrichment score: 1.59; see Figure S2) and Synapse-related proteins (enrichment score: 1.37; see Figure S3). DAVID analysis of our 24 h-LRG-set highlighted a significant regulation of protein kinase and phosphorylation-related genes (enrichment scores 1.98, 2.58 respectively; Figure S4, S5). Closer analysis of the 24 h-LRG-set showed that it contained 39 genes encoding protein kinases, 25 of which were downregulated, as well as 11 phosphatases, 9 of which were downregulated. Thus the DAVID analysis predicts regula- tion of cAMP and G-protein coupled receptor linked pathways 5 h post-LTP that is followed by downregulation of protein kinases/ phosphatases at 24 h. Together these analyses predict that gene expression underpins ongoing modulation of pivotal cell-signalling systems, potentially contributing to a metaplastic shift in calcium dynamics or signalling [25]. Canonical pathway analysis of the 24 h-LRG set identified NFKB (nuclear factor kappa B), Reelin, PTEN (phosphatase and tensin homolog), DHA (omega-3 fatty acid DHA; docosahexaenoic acid) and Clathrin-mediated endocytosis pathways as specifically regulated (Figure 6). Reelin and PTEN signalling are linked to neurogenesis, synaptic plasticity [26], neuronal migration and maturation [27] or local protein synthesis [28]. DHA signalling has previously been linked to LTP [29] and cell survival [30]. Thus these data predict an ongoing role of gene expression supporting synaptic growth and plasticity. Interestingly, PTEN associates with PSD95 and is recruited to the PSD in response to NMDA receptor activation [31] and has been linked to AMPAR trafficking [32]. These data interpreted alongside the observation that Clathrin- mediated endocytosis is a significantly regulated canonical pathway specifically within the 24 h-LRG set suggests that late phase LTP- related gene expression may contribute to homeostatic glutamate receptor regulation. 3. Ingenuity Pathway Analysis: Network analyses To further explore our LRG datasets we used the network analysis tools of the IPA software to predict potential molecular networks functioning during the consolidation of LTP. These analyses revealed both effector pathways and highlight regulatory mechanisms engaged in response to LTP. 3.1 Calcium signalling and LTP persistence. The most significant network formed at 5 h (5 h-Network 1; Figure 7A; Score = 42; direct interactions) included the biological functions Calcium, Protein kinase A and phospholipase C signalling. This supports the primary conclusion from the IPA Canonical pathway and DAVID analysis that regulation of calcium signalling is a key marker of the 5 h-LRG set. Within this network calmodulin forms a central hub that is linked to a type 1 and type 2 calcium/ calmodulin-dependent protein kinases cluster. Interestingly, this network also contains a hub centred on the transcription factor CREB, which interacts with the known LTP-related protein kinase PRKCA (aka PKCa) and its substrate Growth associated protein- 43 (GAP43). Co-regulation of PRKCA and GAP43 is particularly pertinent as phosphorylation of GAP43 and the resulting enhanced release of glutamate have been mooted as a potential mechanism contributing to LTP persistence [33]. As CAMKI phosphorylates and activates synapsin I, a molecule also known to be LTP-regulated [34] and involved in neurotransmitter release, these data suggest a role for gene expression in supporting enhanced release of glutamate 5 h post-LTP, perhaps at downstream mossy fiber terminals in area CA3 [35]. CAMK signalling may contribute to LTP persistence in multiple ways. Recent literature has shown CAMKI-gamma (CAMKIG) to be enriched within the dentate gyrus and linked to dendritogenesis [36,37] and CAMKI-alpha to axonal elongation [38]. As expression of CAMKIG was upregulated in our 5 h-LRG set as well as other molecules linked to postsynaptic reorganisation such as the protein kinase ROCKI (Rho-associated coiled-coil forming protein kinase 1) and its binding partner RHOD, this network may reflect LTP-responsive cytoskeletal reorganisation [39]. Together these data suggest that a key function of the 5 h- LRG network is related to synaptic reorganization. 3.2 Negative regulation of gene expression at 5 h post- LTP. Several molecules involved in negative regulation of gene expression are present in 5 h-Network 1. For example, HistoneH3 forms a central hub and is linked to the epigenetic repressor of transcription HDAC1 (a member of the histone deacetylase complex) and its binding partner RUNX1T1, both of which are upregulated in our 5 h-LRG set. This complex removes acetyl moieties from histones, resulting in condensation of chromatin and transcriptional repression [40]. This network also includes transcriptional repressors such as NUMBL (numb homolog), which acts as a negative regulator of NFKB signalling [41] and HES1 (hairy and enhancer of split 1), which inhibits expression of genes regulated by bHLH (basic helix-loop-helix) proteins [42]. Furthermore, several specific transcriptional activators are down- regulated in our 5 h-LRG set: for example, SMARCA4 (SWI/ PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40538