strain: Fischer 344 × Brown Norway F1 hybrid (F344xBN) Sex: Male age: 7 months tissue: Brain hippocampal region: CA3 drug treatment: VEH behavioral group: RPC
Treatment protocol
One week following standard spatial training (not for home cage controls), subjects used to examine the influence of recent experience on the response to HDACi administration were tested on a one session RPC variant of the Morris water maze, consistting of 17 trials (60 s cutoff), with a 15 s intertrial interval. During trials 1–9, 11, 13, 15, and 17, a black escape platform protruded above the water surface, rendering it visible; and on trials 10, 12, 14, and 16, the platform was hidden, submerged just below the water. A single 60 s probe trial was administered 85 min after the onset of training, immediately before death. Rats were injected with freshly prepared SAHA dissolved in 100% DMSO, administered at 50 mg/kg (final concentration delivered was 50 mg/ml i.p.) per rat, or with an equal volume of 100% DMSO vehicle (Veh). Vehicle and SAHA solutions were administered 10 min before RPC testing, 90 min before death.
Growth protocol
Twenty-eight young (7 months of age) male Fischer 344 × Brown Norway F1 hybrid rats (F344xBN) from the National Institute on Aging colony (Harlan Industries) were singly housed in standard cages in a climate-controlled vivarium maintained on a 12 h light/dark cycle. Food and water were available ad libitum. Subjects acclimated for 1 week and were handled for 5 min/d for a total of 3 d before treatment. Animal procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011) and were approved by the Animal Care and Use Committee of the National Institute on Aging. Standard water maze training was conducted for all rats, except for home cage controls. Briefly, training took place across eight consecutive days, three trials per day (each using a 90 s cutoff), with a 60 s intertrial interval. Every sixth trial was a probe test in which the escape platform was initially retracted to the bottom of the maze for 30 s and then made accessible to permit escape.
Extracted molecule
total RNA
Extraction protocol
Ninety minutes following injection of vehicle or SAHA, animals in all conditions were deeply anesthetized with isoflurane and sacrificed. Brains were removed, and freshly microdissected hippocampal subfields (CA1, CA3, and dentate gyrus [DG]) were frozen and stored at −80°C until RNA extraction using Trizol reagent and further purified using RNEasy mini columns (QIagen, Valencia, CA) according to the manufacturer’s instructions. RNA quantity and integrity was assessed using Nanodrop and Agilent Bioanalyzer RNA 6000 Chip(Agilent, Santa Clara, CA).
Label
Cy3
Label protocol
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5 μg of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
Hybridization protocol
Standard Illumina protocol. In short, a total of 0.75 μg of biotin-labeled cRNA was hybridized at 58°C for 16 hours to Illumina's HumanHT-12 V4.0 Expression Bead Chips (Illumina, San Diego, CA). Each BeadChip has more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
Scan protocol
Arrays were scanned at a resolution of 0.54 μm using the Illumina iScan scanner.
Data processing
Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection scores is supplied.
