Male Sprague-Dawley rats (Charles River Labs, Raleigh, NC, USA), weighing 375 ±25 g, were used in the experiments. Rats were housed in a temperature-controlled (22.2 + 0.2ºC) room with free access to food and water. The animals received a single injection of METH (20 mg/kg) and were euthanized at various time points afterwards. All animal use procedures were according to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee.
Growth protocol
N/A
Extracted molecule
total RNA
Extraction protocol
At the indicated time after the METH or saline injections, rats (n = 5 per group) were euthanized and the NAc was dissected and immediately put on dry ice. The tissues were kept at -70 oC until RNA extraction. Total RNA was isolated from the nucleus accumbens according to the manufacturer’s manual using Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA). RNA integrity was detected using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA).
Label
biotin
Label protocol
N/A
Hybridization protocol
Microarray hybridization was carried out using RatRef-12 Expression BeadChips arrays (22, 523 probes) (Illumina Inc., San Diego, CA) as previously described (Cadet et al., 2010a). In brief, a 600 ng aliquot of total RNA from each NAc sample was amplified using Illumina RNA Amplification kit (Ambion, Austin, TX). Single-stranded RNA (cRNA) was generated and labeled by incorporating biotin-16-UTP (Roche Diagnostics, Indianapolis, IN). 750 ng of each cRNA sample were hybridized to Illumina arrays at 55°C overnight according to the Whole-Genome Gene Expression Protocol for BeadStation (Illumina Inc.). Hybridized biotinylated cRNA was detected with Cyanine3-streptavidin (GE Healthcare, Piscataway, NJ) and quantified using Illumina's BeadStation 500GX Genetic Analysis Systems scanner.
Scan protocol
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina. Quantitative polymerase chain reaction (qPCR): Total RNA was reverse-transcribed to cDNA with oligo dT primer using Advantage RT for PCR kits (BD Biosciences Clontech Laboratories, Palo Alto, CA, USA). Gene-specific primers were generated using Light Cycler Probe Design software and synthesized by the Synthesis and Sequencing Facility of Johns Hopkins University (Baltimore, MD, USA). Real-time PCR experiments were performed using iQ SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) and Chromo 4 Detector (MJ Research, Inc. Waltham, MA. Fluorescence data collection was performed at the end of each extension phase. For all qPCR experiments, individual data were normalized using the corresponding OAZ1 signal as described (McCoy et al., 2011). Western Blot Analysis: NAc tissues were first washed with chilled 0.01 M PBS. Cytoplasmic and nuclear protein fractions were separated using NE-PER nuclear and cytoplasmic extraction reagents based on the manufacturer's instruction (Pierce, Rockford, IL). Protein concentrations were determined with the BioRad Dc Protein assay reagent (BioRad, Temecula, CA). The lysates were then denatured with sample buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromophenol blue, and 50 mM DTT) at 100°C for 5 min, and subjected to SDS-PAGE. Proteins were electrophoretically transferred to Hybond-PTM membrane (Amersham Pharmacia Biotech, Piscataway, NJ) and then incubated with specific antibodies: anti-acetyl-histone H3 (Lys 9) and H3 (Lys 18), anti-acetyl-histone H4 (Lys 8), H4 (Lys 12), and H4 (Lys 16) (Millipore, Billirica, MA 01821, USA); anti-HDAC1 and anti-HDAC2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA 95060, USA); anti-phospho-CREB and anti-ATF2 (Cell Signaling Technology, Danvers, MA 01923, USA). After washing in Tris-buffered saline with 0.1% Tween-20, membranes were pre-incubated in the detergent/blocking buffer containing 1:2000 horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Cell Signaling Technology Inc., Danvers, MA, USA) for 1 hr at room temperature. To confirm equal protein loading, blots were re-probed with α-tubulin antibody (1:4000; Sigma, 2 h at RT). LumiGLO chemiluminescent reagents (Cell Signaling Technology Inc., Danvers, MA, USA) were used to detect protein expression. Signal intensity was measured densitometrically with LabWorks version 4.5 (BioImaging Systems analysis software, BioImaging System, UVP Inc., Upland, CA). For quantification, the signal intensity was normalized using the signal intensity of tubulin.
Description
5130699005_I
Data processing
The Illumina BeadStudio software was used to measure fluorescent hybridization signals. Data were extracted by BeadStudio (Illumina) and analyzed using GeneSpring software v. 7.3.1 (Silicon Genetics, Redwood City, CA). Raw data were imported into GeneSpring and normalized using global normalization. The normalized data were used to identify changes in gene expression at the different time points after the injection of METH. A gene was identified as significantly affected if it showed increased or decreased expression according to an arbitrary cut-off of 1.7-fold changes at p<0.05, according to the GeneSpring statistical package (unpaired t-test). Similar criteria have been successfully used in our previous microarray studies (Cadet et al., 2009: Jayanthi et al., 2009; Cadet et al., 2011). Network analyses were performed using the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA). The IPA software allows for the identification of networks, canonical pathways, and biological pathways that are affected by the drug. We also used the IPA software to graphically show the cellular location of genes significantly affected by METH and to overlay differentially affected networks of interest. Data for quantitative PCR and Western blot analyses are presented as means ± SEM. Statistical analyses were performed using one-way ANOVA analysis followed by Fisher’s protected least significant difference (StatView 4.02, SAS Institute, Cary, NC). Criteria for significance were set at p < 0.05.
Methamphetamine administration causes differential alterations in gene expression and dynamic patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens
