Animals and study design

Six-month-old C57BL/6 J male mice (n = 168 mice in total) were obtained from Jackson Laboratories, Bar Harbor Maine. The biological age of the mice was selected to be relevant to the biological age of astronauts during space missions. The mice were shipped from Jackson Laboratories to Brookhaven National Laboratory (BNL), Upton, New York, and allowed to accommodate to the housing facility there for one week. Subsequently, the mice were irradiated with 0.1, 0.2, or 0.4 Gy of 600 MeV 56Fe or sham-irradiated (n = 42 mice/radiation dose). For irradiation, mice were individually loaded into 8 × 3 × 3 cm plastic square enclosures with air holes and placed in a foam fixture in the beam line of the NASA Space Radiation Laboratory (NSRL). They were exposed to a rectangular beam of approximately 20 x 20 cm. The focused beam of high-energy was generated by the Booster accelerator at BNL and transferred to the experimental beam line at the NSRL facility. Dose calibration was performed so that the desired dose could be delivered. The exposure time for the 0.1 Gy dose was 0.4 min, the exposure time for the 0.2 Gy dose was 0.7 min, and the exposure time for the 0.4 Gy was 1.1 min. Sham-irradiated mice were placed into the plastic enclosures for the same time as the irradiated mice. Mice were randomly assigned to the experimental groups. The week after the irradiation or sham-irradiation, the mice were shipped to Oregon Health & Science University (OHSU) and were assigned to one of two time points (2 or 20 weeks; n = 84 mice/time point). Cognitive testing started two or twenty weeks following irradiation. Following cognitive testing, the mice were killed by cervical dislocation followed by decapitation. The hippocampus of one hemibrain was dissected for DNA methylation and RNA-Seq analyses. The other hemibrain was processed for Arc mRNA and TET2 immunohistochemical analyses. All protocols were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of OHSU and BNL and were in compliance with all Federal regulations.

Novel object recognition

The novel object recognition test was performed as described [42]. The mice were habituated to an open field (16 x 16 in., Kinder Scientific, Poway, CA) for 3 times for 10 min each over three subsequent days. On day 4, the mice were placed in the open field containing two identical objects and they were allowed to freely explore for 15 min. On day 5, the mice were placed again in the open field, but one familiar object was replaced with a novel object. The mice were allowed to explore for 15 min. Movement and time spent exploring each object was recorded using Ethovision XT video tracking system (Noldus Information Technology, Sterling, VA) and hand scored by a researcher blinded to the treatment of the mice. The percent time exploring the novel object, out of the total time exploring the novel and familiar objects on day 5, was used to assess novel object recognition. For each group, the preference for the novel versus the familiar object was assessed. The open field arena and objects were cleaned with 5 % acetic acid between mice and trials.

Water maze

Spatial learning and memory were assessed in the water maze as described [42]. A circular pool (diameter 140 cm) was filled with water made opaque with nontoxic chalk (24 °C) and mice were trained to locate a submerged platform. To determine if irradiation affected the ability to swim or learn the water maze task, mice were first trained to locate a clearly marked platform (visible platform, Days 1 and 2). The mice were subsequently trained to locate the platform when it was hidden beneath the surface of opaque water (Days 3–5). Training during the hidden platform sessions (acquisition) required the mice to learn the location of the hidden platform based on extra-maze cues. For both visible and hidden sessions, there were two daily sessions, morning and afternoon, which were 2-h apart. Each session consisted of two trials (with 5-min inter-trial intervals). A trial ended when the mice located the platform. Mice that failed to locate the platform within 60 s were led to the platform by placing a finger in front of their swim path. The mice were taken out of the pool after they were physically on the platform for a minimum of 3 s. During visible platform training, the platform was moved to a different quadrant of the pool for each session. For the hidden platform training, the platform location was kept constant. The mice were placed into the water facing the edge of the pool in one of nine randomized locations. The start location was changed for each trial. The swimming patterns of the mice were recorded with Noldus Ethovision video tracking software (Ethovision XT, Noldus Information Technology, Wageningen, Netherlands) set at six samples/s. The time to locate the platform (latency) and cumulative distance to the target were used as measures of performance for the visible and hidden sessions. Because swim speeds can influence the time it takes to reach the platform, they were also analyzed.

To measure spatial memory retention, probe trials (platform removed) were conducted 1 h after the last hidden trial of each mouse on the first and second day of hidden platform training and 72 h following the last hidden trial of each mouse on the third day of hidden platform training and the cumulative distance to the target location during the probe trials was analyzed.

Hippocampal network stability

Three days after the last water maze test day, exploration of identical or different environments was used to study the stability of hippocampal networks [27, 31]. The method called catFISH (cellular compartment analysis of temporal activity using fluorescence in situ hybridization) relies on the precise temporal kinetics of the IEG Arc, which has been used to provide important insight into the post-transcriptional infrastructure of gene expression involved in synaptic plasticity and memory [27, 31], (for review, [28]). When neurons are engaged in information processing, Arc is rapidly transcribed and can be visualized and quantified after ~5 min. Subsequently, the mRNA is translocated to the cytoplasm where it remains detectable for ~20-30 min after the initial transcription. Ultimately, the mRNA is translocated to tagged synapses for protein synthesis. Thus, two different cellular compartments (nuclear and cytoplasmic) can be clearly distinguished, providing means to identify which neurons were active during distinct behavioral experiences [31].

Eight mice from each experimental radiation condition were placed individually into a novel environment (A) and allowed to explore for five minutes, as described [31]. Environment A is a square open field (61 × 61 cm box with 20-cm high walls). After exploration, mice were returned to their cage for 25 min, returned to the same environment for an additional 5 min (AA Paradigm). Another 8 mice of each experimental radiation condition explored environment A for 5 min, and 25 min later they were placed in a different environment (B), a circular arena 45 cm in diameter, and allowed to explore for 5 min (AB Paradigm). Following the last environmental exposure, the mice were killed by cervical dislocation and the brains quickly removed, as described above.

Using catFISH, Arc mRNA appear as discrete nuclear foci (recent transcription ~5-10 min), and/or as diffuse mRNA in the cytoplasm (earlier transcription). Nuclear and cytoplasmic Arc can be distinguished using intronic and full-length probe respectively labeled with digoxigenin or fluorescein. Arc staining was classified as: a) None (no Arc staining); b) cytoplasmic Arc staining only; c) nuclear Arc staining only; or d) both Arc-foci/Arc-cyto (containing both foci and cytoplasmic staining). We can determine if the neurons responding (i.e. Arc+) to an initial experience (exploration of environment A) are the same neurons that respond to a second and identical experience as expected based on sham-irradiated animals (AA Paradigm), and if the hippocampal networks activated during exploration of the first environment are statistically independent from those activated by the exploration of a different environment (AB Paradigm). For the CA1 and CA3 regions of the hippocampus, the percentage of neurons showing both foci and cytoplasmic staining was analyzed. As the pattern of percentages of Arc-positive neurons in both environments might reflect differences in the total number of Arc-positive neurons (total number of neurons with Arc nuclear foci, cytosolic Arc, or both) this number was also determined. For the dentate gyrus, the percent of neurons expressing Arc only in the cytoplasm or expressing both Arc-foci/Arc-cyto (containing both foci and cytoplasmic staining) was analyzed. This measure was analyzed as it reflects the total response to the environment(s).

Tet2 Immunohistochemistry

Following cervical dislocation and decapitation, hemibrains were rapidly removed and frozen using isopentane and dry ice, as described [26] and stored at −80 °C. The hemibrains were shipped to the University of California, San Francisco, and cut at 20 μm using a cryostat. Slides were sent to OHSU for analysis of Tet2 immunoreacttivity using a specific primary antibody from Santa Cruz Biotechnology (Tet2 S-13, catalog number sc-136926). Briefly, sections (n = 3 sections per hemibrain and approximately 200 μm apart) were rinsed in phosphate buffered saline (PBS), and incubated in 4 % normal goat serum (NGS) in PBS with 0.4 % triton X-100 (PBS-TX). Next, sections were incubated overnight with primary antibodies against Tet2 (1:250) in 4 % NGS in PBS-TX. The next day, tissue sections were washed in PBS, incubated for 2.5 h in donkey anti-rabbit Alexa 488 (1:200) in 4 % NGS in PBS-TX and again rinsed in PBS. Analysis of Tet2 immunoreactivity was performed using an Olympus IX81 confocal microscope equipped with Slidebook software. Images of hippocampal regions (CA1, CA3, and dentate gyrus) and the cortex were captured within 3 sections (Bregma −1.58 to −2.46) using a 20x objective (UPlan FL, Olympus). Tet2 immunoreactivity was quantified within fixed area frames; CA1 (box, 125 × 50 μm), CA3 (2 boxes, 95 × 85 μ m each), dentate gyrus (2 boxes, 95 × 85 μm each), and cortex (posterior parietal association area; box, 240 × 200 μm). Background threshold levels were set and applied to all images for comparison. Pixel intensities above this threshold were used for quantification measures (area occupied by pixels and intensities of pixels). The total intensity was also quantified as a measure of overall pixel intensity within a specific brain region.

DNA methylation

DNA was isolated from the hippocampus. Antibodies against 5mC and 5hmC were used to immunoprecipitate sonicated DNA preparations for methyl-DNA immunoprecipitation (meDIP) and hydroxymethyl-DNA immunoprecipitation (hmeDIP, respectively, from twelve pools of tissues (6 x 2 pools of hippocampal tissues or 2 pools/radiation condition/time point). These antibodies were used to precipitate genomic regions that are enriched for either 5mC or 5hmC. Following immunoprecipitation, high-throughput genomic sequencing and segmentation analyses were used to identify enriched genomic regions. For DIP-Seq library preparation, RNAse-treated DNA was isolated using the Qiagen Allprep DNA/RNA protocol. The DNA was sonicated using a Cole Parmer CPX-132 sonicator (75 % amplitude, 3×10’) and polished using the DNA terminator end repair kit (Lucigen). DNA fragments were A-tailed using Klenow exo- (Epicenter) and ligated to un-methylated HT TrueSeq indexed adapters and purified. The resulting purified DNA was denatured at 95 °C, resuspended in 100 ul of DIP IP buffer, and immunoprecipitated with 1 μg of the highly specific 5-methylcytosine antibody (EMD Millipore) or 2 ul of 5-hydroxymethylcytosine (Active Motif) antibody and Dynal anti-mouse IgG beads. Beads were rinsed 7 times with IP buffer, eluted with 1 % SDS at room temperature and the eluted DNA purified and subjected to limited amplification (~18 cycles). Libraries were sequenced on the HiSeq2000 platform at the OHSU Massively Parallel Sequencing Shared Resource or the Oregon State University Center for Genome ReseArch. DIP-Seq regions methylated above “background” were identified using an optimized sliding window parameter and enriched regions selected over background models via a Montecarlo-permutation test [43].

RNA-Seq

To facilitate direct comparison of DIP-Seq data with gene expression data, RNA-Seq was used to profile transcription from the same animals used for the DIP-Seq experiments. RNA was isolated using the NEBnext poly A selection kit (New England Biolabs). Illumina high-throughput sequencing technology was used to profile RNA levels in an unbiased manner. Differential methylation may occur at novel regions and the unbiased nature of RNA-Seq analyses enables analysis of associated un-annotated transcription. For Illumina RNA-Seq library preparation, we used the NEBnext Ultra kit according to the manufacturers specifications (New England Biolabs).

Illumina data were mapped to the UC Santa Cruz assembly using Bowtie [44]. For RNA-Seq analyses, tags that overlap with known RefSeq (UCSC RefSeq annotation) were counted using R scripts [45]. Significance was assessed using the DESeq2 package [46]. The Storey Q-test was used to adjust for multiple comparisons [47].

Libraries were sequenced on the HiSeq2000 platform at the OHSU Massively Parallel Sequencing Shared Resource.

Real time PCR

For real-time PCR, 20–22 bp primers were designed using MIT's Primer3 software (http://bioinfo.ut.ee/primer3/) using standard parameters and all quantitation utilized standard curve real-time PCR. Primers will be provided upon request. PCRs (10 μl) contained 1 μl 10× PCR buffer (Invitrogen), 2.5 mM MgCl 2 , 200 μM dNTP (Roche), 0.125–0.25 μM primer (IDT), 1× SYBR green I (Invitrogen), and 1 U platinum Taq (Invitrogen). PCR was run on an Opticon OP346 (MJ Research) for one cycle at 95 °C, 35 s, and 30–50 cycles at 94 °C, 15 s; 68 °C–70 °C, 40 s. RT-PCR experiments were normalized to 18S RNA levels (other housekeeping genes showed similar results) and were expressed as ng of gel-purified (Qiagen) amplicon.

Bioinformatics and statistical analyses

All behavioral, cognitive, and Arc data are shown as mean ± SEM. The statistical analyses of the data were performed using SPSS™ (Chicago, IL) and GraphPad Prism™ (San Diego, CA) software packages. To compare effects of radiation across groups, ANOVA was used with irradiation as the between factor, followed up by Dunnett’s posthoc tests or t-tests when appropriate. To analyze locomotor activity over three days, repeated measures ANOVA was used. To assess effects of radiation on Arc measures across dose conditions in mice exposed to a specific environmental condition, one-way ANOVAs were used. To compare exploration of the objects and the percentage of Arc-positive cells and total number of Arc cells following exposure to the two different environmental conditions, 2-sided t-tests were used. All figures were generated using GraphPad Prism software. We considered p < 0.05 as statistically significant.

Single read sequence data was mapped to the mouse reference genome (UCSC mm9) using the Bowtie algorithm using standard flags except for allowing 2 mismatches [44]. Sequences that map to a single location were selected and domains enriched for 5mC or 5hmC were selected using a parameter-optimized Monte-Carlo-based segmentation algorithm [43]. A 1000 bp sliding-window was selected based on iterative analyses that maximized the number of enriched regions. A comparison of different high-throughput sequencing based methods to study DNA methylation concluded that MeDIP-Seq covers ~ 67 % of genomic CpGs [48].

For statistical comparisons of biological samples, regions of methylation enrichment were merged and differences in methylation interrogated with FDR-adjusted chi-square or negative binomial statistics [49]. Statistical and visualization studies involved the R programming language and Bioconductor packages [49]. Gene ontology analyses utilized the Bioconductor Goseq package, which adjusts for RNA-Seq length bias artifacts [50]. For gene ontology analyses the top 2000 DMRs (differentially methylation regions) or DHRs (differentially hydroxymethylated regions) (FDR-adjusted p < 0.01) within 50 kb of a transcriptional start site were non-redundantly annotated. Unless otherwise stated, overlap between DMRs and RNA-Seq data was analyzed using a similar relational approach.

Pathway analyses involved standard bioconductor packages (e.g. cmap, keggraph, gsea). DIP sequence-tag heatmaps were generated in R by plotting median-normalized DIP-Seq tag density in gene bodies and indicated flanking regions with color-maps scaled to the 80 % quantile. Statistical analyses of pathway data were conducted via FDR-adjusted Fisher exact or KS-tests. We considered (FDR-adjusted) p < 0.01 as statistically significant.