Research Articles: Cellular/Molecular Distinct transcriptomic profiles in the dorsal hippocampus and prelimbic cortex are transiently regulated following episodic learning https://doi.org/10.1523/JNEUROSCI.1557-20.2021 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.1557-20.2021 Received: 19 June 2020 Revised: 25 November 2020 Accepted: 6 January 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 the authors 1 Distinct transcriptomic profiles in the dorsal hippocampus and prelimbic cortex are 2 transiently regulated following episodic learning 3 Abbreviated Title: Distinct dHC and PL transcriptomes after learning 4 5 Aaron Katzman1, Alireza Khodadadi-Jamayran2, Dana Kapeller-Libermann1, Xiaojing Ye1, 6 Aristotelis Tsirigos2, Adriana Heguy3 and Cristina M. Alberini1,4 7 8 1 Center for Neural Science, New York University, New York, NY, 10003, USA 9 2 Applied Bioinformatics Laboratories, NYU Grossman School of Medicine, New York, NY 10 10016, USA 11 3 Genome Technology Center and Department of Pathology, NYU Grossman School of 12 Medicine, New York, NY 10016, USA 13 4 Correspondence should be addressed to: 14 Cristina M. Alberini 15 Center for Neural Science, New York University, 4 Washington Place, Room 809, New York, 16 NY, 10003; Email: [email protected]; Phone: 212-998-7721 17 18 Number of pages: 52 19 20 Acknowledgments: The studies presented in this manuscript were supported by NIH grant 21 MH065635 to CMA. AK was supported by F31MH116585. The RNA-seq analysis was carried 22 out using High Performance Computing resources at NYU Langone Health and library 23 preparation and sequencing through the Genome Technology Center (GTC). This shared 24 resource is partially supported by the Cancer Center Support Grant, P30CA016087, at the Laura 25 and Isaac Perlmutter Cancer Center. Current affiliation of XY: Forensic Medicine, Zhongshan 26 School of Medicine, Sun Yat-sen University, Guangzhou, China. 27 28 Conflicts of interest: The authors declare no competing financial interests 2 29 Abstract 30 A fundamental, evolutionarily conserved biological mechanism required for long-term memory 31 formation is rapid induction of gene transcription upon learning in relevant brain areas. For 32 episodic types of memories, two regions undergoing this transcription are the dorsal 33 hippocampus (dHC) and prelimbic (PL) cortex. Whether and to what extent these regions 34 regulate similar or distinct transcriptomic profiles upon learning remains to be understood. Here, 35 we used RNA sequencing in the dHC and PL cortex of male rats to profile their transcriptomes 36 in untrained conditions (baseline) and at 1 hour and 6 days after inhibitory avoidance learning. 37 We found that, out of 33,713 transcripts, over 14,000 were significantly expressed at baseline in 38 both regions and approximately 3,000 were selectively enriched in each region. Gene Ontology 39 biological pathway analyses indicated that commonly expressed pathways included synapse 40 organization, regulation of membrane potential, and vesicle localization. The enriched pathways 41 in the dHC were gliogenesis, axon development, and lipid modification, while in the PL cortex 42 included vesicle localization and synaptic vesicle cycle. At 1 hour after learning, 135 transcripts 43 changed significantly in the dHC and 478 in the PL cortex; of these, only 34 were shared. 44 Biological pathways most significantly regulated by learning in the dHC were protein 45 dephosphorylation, glycogen and glucan metabolism, while in the PL cortex were axon 46 development and axonogenesis. The transcriptome profiles returned to baseline by 6 days after 47 training. Thus, a significant portion of dHC and PL cortex transcriptomic profiles is divergent 48 and their regulation upon learning is largely distinct and transient. 49 3 50 Significance Statement 51 Long-term episodic memory formation requires gene transcription in several brain regions 52 including the hippocampus and prefrontal cortex. The comprehensive profiles of the dynamic 53 mRNA changes that occur in these regions following learning are not well understood. Here, we 54 performed RNA sequencing in the dorsal hippocampus (dHC) and prelimbic (PL) cortex, a 55 prefrontal cortex subregion, at baseline, 1 hour, and 6 days after episodic learning in rats. We 56 found that at baseline, dHC and PL cortex differentially express a significant portion of mRNAs. 57 Moreover, learning produces a transient regulation of region-specific profiles of mRNA, 58 indicating that unique biological programs in different brain regions underlie memory formation. 4 59 Introduction 60 A fundamental and evolutionarily conserved mechanism required for the formation of long-term 61 memory is an initial phase of de novo transcription and translation in the relevant nervous system 62 regions (Klann and Dever, 2004; Costa-Mattioli et al., 2009; Alberini and Kandel, 2014). These 63 processes are necessary for stabilizing a labile memory representation, a process known as 64 memory consolidation (McGaugh, 2000; Dudai, 2012; Squire et al., 2015). 65 For episodic types of memories, which store spatial and contextual information, de novo 66 transcription required for memory consolidation occurs in the medial-temporal lobe regions, 67 which include the hippocampus (HC) as well as functionally linked cortical regions such as the 68 prefrontal cortex (PFC) (Dash et al., 2004; Wiltgen et al., 2004; Alberini, 2009). Over time, the 69 HC appears to become dispensable for long-term memory storage, while cortical regions remain 70 functionally engaged, suggesting that distinctive regional biological mechanisms must be 71 responsible for memory consolidation. This temporal redistribution of the memory 72 representation, which is critical for maintaining and storing the learned information long term, is 73 known as systems-level consolidation (Frankland and Bontempi, 2005; Goshen et al., 2011; 74 Dudai et al., 2015; Squire et al., 2015). 75 To fully understand how long-term memories are formed and stored we need a 76 comprehensive understanding of the transcriptomic profiles that change in response to learning 77 across the brain regions that constitute the activated memory system, as well as of their 78 progression over time. In other words, we need to obtain a system(s) biology understanding of 79 memory, which is still in large part lacking. Furthermore, for decades, the molecular studies on 80 long-term memory consolidation focused mostly on the characterization of mechanisms in single 81 brain regions, particularly the HC. Only more recently has some attention been turned to cortical 5 82 regions. Comprehensive, unbiased biological assessments such as RNA sequencing (RNAseq) 83 have been carried out but have been mostly limited to single brain regions. For example, Cho et 84 al. (2015; also see Cho et al. 2016; Mathew et al. 2016) employed RNAseq together with 85 ribosomal profiling (active translational profiles) in the mouse HC after contextual fear 86 conditioning and found an initial translational wave at 5 minutes after training followed by 87 multiple transcriptional waves, beginning 10-30 minutes after training and continuing for 4 88 hours. Bero et al. (2014) performed RNAseq in the anterior cingulate cortex (ACC), a subregion 89 of the PFC involved in memory storage, at 1 hour after contextual fear conditioning in mice and 90 reported a significant upregulation of synaptic plasticity transcriptional profiles paired with 91 dendritic spine remodeling. Rizzo et al. (2017), using RNAseq on polyribosomal-associated 92 mRNAs, reported two distinct waves of mRNA recruitment to actively translating ribosomes in 93 the PFC (ACC, PL, and infralimbic (IL) cortices) at 1 and 6 hours after contextual fear learning 94 in mice. These and other previous studies emphasized the general concept that brain plasticity 95 mechanisms underlie memory formation. In most cases, these studies focused their attention on 96 the fundamental pathways shared across brain regions and systems; however, whether distinct 97 mechanisms are also engaged in each brain region remains to be determined. Halder et al. (2016) 98 employed RNAseq, chromatin immunoprecipitation (ChIP)-seq, and methylated DNA 99 immunoprecipitation (MeDIP)-seq in the hippocampal CA1 subregion and ACC after contextual 100 fear conditioning in mice at 1 hour and 4 weeks after training. They reported changes in 101 transcription and methylation profiles of plasticity genes in both CA1 and ACC at 1 hour after 102 training and differential methylation changes only in the ACC at 4 weeks post-training (Halder et 103 al., 2016). Other studies compared cell type-specific transcriptional profiles in different regions 104 such as the HC and PFC in mice, but only at baseline (Zhang et al., 2014; Zeisel et al., 2015; 6 105 Cembrowski et al., 2016). These results provided important cell type-specific transcriptional 106 databases, but with the caveat that cell sorting techniques damage cytoplasmic and peripheral 107 cell compartments (van den Brink et al., 2017; Nguyen et al., 2018) where rapid mRNA changes 108 occur in response to learning (Miyashiro et al., 1994; Zhong et al., 2006; Ostroff et al., 2019).