DOI: 10.25338/b8qs4f
title: Dataset for the transcriptome analysis of hippocampal subfields identifies gene expression profiles associated with long-term active place avoidance memory

description: The hippocampus plays a critical role in storing and retrieving spatial
 information. By targeting the dorsal hippocampus and manipulating specific
 “candidate” molecules using pharmacological and genetic manipulations, we
 have previously discovered that long-term active place avoidance memory
 requires transient activation of particular molecules in dorsal
 hippocampus. These molecules include amongst others, the persistent
 kinases Ca-calmodulin kinase II (CaMKII) and the atypical protein kinase C
 isoform PKC iota/lambda for acquisition of the conditioned behavior,
 whereas persistent activation of the other atypical PKC, protein kinase M
 zeta (PKM zeta) is necessary for maintaining the memory for at least a
 month. It nonetheless remains unclear what other molecules and their
 interactions maintain active place avoidance long-term memory, and the
 candidate molecule approach is both impractical and inadequate to identify
 new candidates since there are so many to survey. Here we use a
 complementary approach to identify candidates by transcriptional profiling
 of hippocampus subregions after formation of the long-term active place
 avoidance memory. Interestingly, 24-h after conditioning and soon after
 expressing memory retention, immediate early genes were upregulated in the
 dentate gyrus but not Ammon’s horn of the memory expressing group. In
 addition to determining what genes are differentially regulated during
 memory maintenance, we performed an integrative, unbiased survey of the
 genes with expression levels that covary with behavioral measures of
 active place avoidance memory persistence. Gene Ontology analysis of the
 most differentially expressed genes shows that active place avoidance
 memory is associated with activation of transcription and synaptic
 differentiation in dentate gyrus but not CA3 or CA1, whereas
 hypothesis-driven candidate molecule analyses identified insignificant
 changes in the expression of many LTP-associated molecules in the various
 hippocampal subfields, nor did they covary with active place avoidance
 memory expression, ruling out strong transcriptional regulation but not
 translational regulation, which was not investigated. These findings and
 the data set establish an unbiased resource to screen for molecules and
 evaluate hypotheses for the molecular components of a
 hippocampus-dependent, long-term active place avoidance memory.
description: All animal
 care and use comply with the Public Health Service Policy on Humane Care
 and Use of Laboratory Animals and were approved by the New York University
 Animal Welfare Committee and the Marine Biological Laboratory IACUC. Male
 C57BL/6J mice were housed at the Marine Biological Laboratory on a 12:12
 (light: dark) cycle with continuous access to food and water in home cages
 with up to five littermates. To examine spatial
 learning and memory, we used a well-established active place avoidance
 paradigm. Littermates were randomly assigned to one of our treatment
 groups (standard-trained, n=8; standard-yoked, n=8; conflict-trained, n=9;
 conflict-yoked, n=9. All mice were exposed to nine 10-min trials in the
 active place avoidance arena. Mice were placed on an elevated circular
 40-cm diameter arena made of parallel bars that rotated at 1 rpm. The
 arena wall was transparent and thus contained the mouse on the arena while
 allowing it to observe the environment. The location of the mouse in the
 arena was determined from an overhead digital video camera interfaced to a
 PC-controlled tracking system (Tracker, Bio-Signal Group Inc., Acton, MA).
 Trained mice in the active place avoidance task are conditioned to avoid
 the location of mild shocks (constant current 0.2 mA, 500 ms, 60 Hz) that
 can be localized by visual cues in the environment. Yoked-control mice are
 delivered the identical sequence of shocks that was received by a
 particular trained mouse, the difference being that for the yoked mice,
 the shocks cannot be avoided or localized to a portion of the environment.
 Mice are allowed to become familiar with walking on the rotating arena
 during a pretraining trial with no shock. Then each mouse received three
 training trails separated by a 2-h inter-trial interval. The mice were
 returned to their home cage overnight. The next day, each mouse received a
 “Retest trial” with the shock in the same location as before. For the next
 three training trials, the shock zone remains in the same place for
 standard-trained animals but is relocated 180<sup>°</sup> for
 the conflict-trained mice. The next day, all mice receive a memory
 “Retention trial” with the shock off to evaluate the strength of the
 conditioned avoidance. A day after the last training
 session, and 30 minutes after the retention session without shock, mice
 were anesthetized with 2% (vol/vol) isoflurane for 2 minutes and
 decapitated. Transverse 300 μm brain slices were cut using a vibratome
 (model VT1000 S, Leica Biosystems, Buffalo Grove, IL) and incubated at
 36°C for 30 min and then at room temperature for 60-90 min in oxygenated
 artificial cerebrospinal fluid (aCSF in mM: 125 NaCl, 2.5 KCl, 1 MgSO4, 2
 CaCl2, 25 NaHCO3, 1.25 NaH2PO4, and 25 Glucose). Slices were cut in half
 so that one hemisphere could be used for RNA-seq and one for ex vivo slide
 physiology. For RNA-sequencing, the DG, CA3, CA1
 subfields were micro-dissected using a 0.25 mm punch (Electron Microscopy
 Systems) and a Zeiss dissecting scope. RNA was isolated using the Maxwell
 16 LEV RNA Isolation Kit (Promega). RNA libraries were prepared by the
 Genomic Sequencing and Analysis Facility at the University of Texas at
 Austin and sequenced on the Illumina HiSeq platform. Reads were processed
 on the Stampede Cluster at the Texas Advanced Computing Facility. Quality
 of raw and filtered reads was checked using the program FASTQC (Wingett
 and Andrews, 2018) and visualized using MultiQC (Ewels <i>et
 al.</i>, 2016). We obtained 6.9 million ± 6.3 million reads per
 sample. Next, we used Kallisto to pseudo-align raw reads to a mouse
 references transcriptome (Gencode version 7), which yielded 2.6 million ±
 2.1 million reads per sample. Mapping efficiency was about 42%. Transcript
 counts from Kallisto were imported into R  and aggregated to yield gene
 counts using the gene identifier from the Gencode transcriptome. DESeq2
 was used to normalize and quantify gene expression with a false discovery
 corrected (FDR) p-value &lt; 0.1. ShinyGo was used to identify Gene
 Ontology terms associated with genes that are correlated with PC1. All
 genes associated with particular GO terms were identified using the Gene
 Ontology Browser . We compared the GO terms for candidate genes,
 differentially expressed genes, and a list of genes identified as
 important for long-term potentiation. We relied on the R packages ggplot2,
 cowplot, and corrr for data visualization.  Spatial
 behavior was evaluated by automatically computing (TrackAnalysis software
 (Bio-Signal Group Corp., Acton, MA) 26 measures that characterize a
 mouse’s use of space during the trial. All statistical analyses were
 performed using R version 3.6.0 (2019-04-26) -- "Planting of a Tree”,
 relying heavily on the software from the tidyverse library. Principal
 component analysis (PCA) was conducted to reduce the dimensionality of the
 data. One- and two-way ANOVAs were used to identify group differences in
 behavioral measures across one or multiple trials, respectively. For
 statistical analysis of gene expression, we used DESeq2 to normalize and
 quantify gene counts with a false discovery corrected (FDR) p-value
 &lt; 0.1. DESeq2 models evaluated gene expression differences either
 between the four behavioral treatment groups (standard-trained,
 standard-yoked, conflict-trained, and conflict-yoked) or between the
 combined memory-trained and combined yoked-control groups.
 Raw sequence data and differential gene expression data are
 available in NCBI's Gene Expression Omnibus Database (accession: GSE99765).