Temporal Profiling of Gene Networks Associated with the Late Phase of Long-Term Potentiation In Vivo Margaret M. Ryan1,2,3, Brigid Ryan1,2, Madeleine Kyrke-Smith1,2, Barbara Logan1,4, Warren P. Tate1,3, Wickliffe C. Abraham1,4, Joanna M. Williams1,2* 1 Brain Health Research Centre, University of Otago, Dunedin, New Zealand, 2 Department of Anatomy, Otago School of Medical Sciences, Dunedin, New Zealand, 3 Department of Biochemistry, Otago School of Medical Sciences, Dunedin, New Zealand, 4 Department of Psychology, University of Otago, Dunedin, New Zealand Abstract Long-term potentiation (LTP) is widely accepted as a cellular mechanism underlying memory processes. It is well established that LTP persistence is strongly dependent on activation of constitutive and inducible transcription factors, but there is limited information regarding the downstream gene networks and controlling elements that coalesce to stabilise LTP. To identify these gene networks, we used Affymetrix RAT230.2 microarrays to detect genes regulated 5 h and 24 h (n = 5) after LTP induction at perforant path synapses in the dentate gyrus of awake adult rats. The functional relationships of the differentially expressed genes were examined using DAVID and Ingenuity Pathway Analysis, and compared with our previous data derived 20 min post-LTP induction in vivo. This analysis showed that LTP-related genes are predominantly upregulated at 5 h but that there is pronounced downregulation of gene expression at 24 h after LTP induction. Analysis of the structure of the networks and canonical pathways predicted a regulation of calcium dynamics via G-protein coupled receptors, dendritogenesis and neurogenesis at the 5 h time-point. By 24 h neurotrophin-NFKB driven pathways of neuronal growth were identified. The temporal shift in gene expression appears to be mediated by regulation of protein synthesis, ubiquitination and time-dependent regulation of specific microRNA and histone deacetylase expression. Together this programme of genomic responses, marked by both homeostatic and growth pathways, is likely to be critical for the consolidation of LTP in vivo. Citation: Ryan MM, Ryan B, Kyrke-Smith M, Logan B, Tate WP, et al. (2012) Temporal Profiling of Gene Networks Associated with the Late Phase of Long-Term Potentiation In Vivo. PLoS ONE 7(7): e40538. doi:10.1371/journal.pone.0040538 Editor: Valerie W. Hu, The George Washington University, United States of America Received December 1, 2011; Accepted June 8, 2012; Published July 10, 2012 Copyright: 2012 Ryan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The Royal Society of New Zealand Marsden Fund; Grant number 03-UOO-118. University of Otago Grants Committee. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: joanna.williams@anatomy.otago.ac.nz Introduction Long-term potentiation (LTP) is widely regarded as a memory storage mechanism as it fulfils key properties of a mnemonic device. For example, LTP is specific to only closely grouped synapses activated following high-frequency stimulation [1,2]; LTP shows both cooperativity and associativity, such that for LTP induction a threshold intensity is required, as is coincident activity of the pre- and postsynaptic neurons. LTP is also remarkably persistent, when induced at perforant path synapses of awake freely moving animals, LTP has been shown to transition through distinct phases which culminate in a potentiation that can remain stable for months and in one documented case, even for a year [3]. The molecular mechanisms underlying the early events have been thoroughly characterised both in vitro and in vivo. Protein kinase activation and subsequent trafficking of AMPA-glutamate recep- tors to the cell surface, together with altered release of neurotransmitter [4], underpin the early phase of LTP. This is followed by trafficking of NMDA-glutamate receptors [5] and metabotropic glutamate receptors [6] that facilitate a change in the calcium dynamics of activated synapses and reflect metaplas- ticity events [7]. There is considerable evidence that late phases of LTP (LTP2 and LTP3) require new protein synthesis, both rapidly via regulated translation of synaptically located mRNA and over the long-term via new gene transcription [8]. The role that new gene expression plays in LTP has been debated, however, and there is evidence that LTP can persist under certain in vitro conditions without protein synthesis [9,10,11]. LTP-related gene expression might contribute indirectly to replenish proteins depleted in response to synaptic activity, while LTP itself is maintained by enzymatic storage mechanisms [12]. Nonetheless, evidence primarily from studies of LTP in vivo strongly supports the hypothesis that new gene expression is crucial for the persistence of LTP. General inhibitors of transcription and translation or knockdown of specific LTP-regulated genes inhibit the mainte- nance of LTP in vivo, and clusters of genes have been identified as regulated by LTP-inducing stimulation [13,14,15]. Of these genes, those that rapidly respond fall into two categories: inducible transcription factors (e.g. members of the EGR and AP1 families) and effector genes (e.g. ARC and HOMER1A), suggesting that new gene expression contributes to LTP maintenance by different modalities. However, the temporal organization of the LTP- related late-gene expression, as driven by the constitutive and inducible transcription factors, has yet to be determined. Our proposal is that to fully understand the complex molecular mechanisms underlying LTP, it is essential to carry out genome- wide analyses to identify and interpret the gene networks regulated PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40538