Comment[ArrayExpressAccession] E-GEOD-51730 MAGE-TAB Version 1.1 Public Release Date 2013-10-28 Investigation Title Profiling the transcriptome: synaptoneurosomes capture the molecular effects of alcohol consumption Comment[Submitted Name] Profiling the transcriptome: synaptoneurosomes capture the molecular effects of alcohol consumption Experiment Description Action of alcohol on synaptic mRNA in the amygdala of mice Chronic alcohol consumption induces changes in gene expression, causing persistent long-term neuro-adaptations and the remodeling of synaptic structures. These alcohol-induced synaptic changes may rely specifically on the local translation of mRNAs in the synaptic compartments of the cell. We profiled the transcriptome from synaptoneurosomes (SN) and paired total homogenates (TH) of amygdala to analyze the synaptic adaptations induced by chronic voluntary alcohol consumption in mice. In the SN both the number of alcohol-responsive mRNAs and the magnitude of fold-change were greater than in the TH. Accordingly, the SN detected many genes with coordinated patterns of expression producing a highly connected mRNA network in gene co-expression analysis. The greater sensitivity of the SN preparation allowed for improved cell-type specificity analysis, revealing an up-regulation of alcohol-responsive astrocytic and microglial modules that correlated with alcohol consumption. Alcohol was found to induce changes in the SN functionally important biological pathways, including long-term potentiation, long-term potentiation depression, glutamate pathway, neuro-immune, RNA-processing and translational machineries and provided overlap with changes seen in human alcoholic brain. Transposable elements were responsive to alcohol and found in the down-regulated neuronal mRNA module, which may underlie some of the coordinated gene expression changes associated with alcohol. We provide evidence that enrichment of synaptic components reveals a more intricate network of coordinated gene expression. Increased resolution captures the molecular effects of synaptic manipulations and provides an improved technique for identifying therapeutic targets for alcohol abuse. Synaptoneurosomes (SN) and Paired total homogenates (TH) were prepared from the same homogenate. 42 microarrays were used in total for the alcohol-control analysis (8 alcohol treated mice and 13 controls), 21 SN and 21 paired TH. 2 TH (control) samples were found outliers and were removed from the analysis. For the SN vs. TH analysis, the 2 TH samples found outliers were removed with the 2 paired SN samples. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Moat Most Ferguson Blednov Mayfield Harris Person First Name Dana Dana Laura Yuri R R Person Mid Initials D A Person Email danamost@gmail.com Person Affiliation University of Texas at Austin Person Address Waggoner center for alcohol and addiction research, University of Texas at Austin, 2500 Speedway, MBB 1.138, Austin, Texas, USA Person Roles submitter Protocol Name P-GSE51730-4 P-GSE51730-5 P-GSE51730-1 P-GSE51730-2 P-GSE51730-3 P-GSE51730-6 Protocol Description RNA samples were processed at the University of Texas Southwestern Medical Center microarray facility in Dallas. mRNA was amplified and biotin-labeled using the Illumina TotalPrep RNA Amplification kit (Ambion, TX) and hybridized to Mouse WG-6 v2.0 Expression BeadChips (Illumina, CA). Each array contained SN and paired TH samples from control and alcohol-treated mice. These were assigned randomly to each array. According to manufacturerM-bM-^@M-^Ys instructions. Mice were given a 24-hour/day access to a two-bottle choice protocol using bottles containing water and/or 20% ethanol and the volume consumed from both bottles was recorded daily as described by (Osterndorff-Kahanek, Ponomarev et al. 2013). Bottle positions were changed daily to control for position preferences. All procedures were approved by the Institutional Animal Care and Use Committee and adhere to NIH Guidelines. The brains were removed and washed for one minute with 1ml of ice-cold-Homogenizing Buffer (HB) containing Hepes [20mM], EDTA [1mM](pH 7.4), RNAseOut, phosphatase and protease inhibitors (from Invitrogen, Sigma, Roche, respectively). Brains were then placed in a coronal Zivic-mouse-brain-slicer (previously washed with HB) with a 0.5mm resolution (Zivic Instruments, PA) and sliced in the following coordinates in order to isolate extended amygdala-containing slices: coronal level 56-66 [Bregma (-0.18)-(-1.155)] and 66-80 [Bregma (-1.155)-(-2.55)]. The extended amygdala was dissected and placed in a tube containing ice-cold HB (250) and homogenized using a VWR homogenizer. To minimize homogenate loss, the pestles were washed with 50ml HB after use and the wash was collected and added to the sample. 10% of the homogenate (30ml) was snap frozen in liquid nitrogen and stored at -80M-0C for RNA total homogenate (TH) analysis. The rest of the homogenate (270ml) was filtered with a 100um-pore filter and a 5um-pore filter (Millipore) (filters were washed with HB before use for protection from RNAse). To maximize yield, the filters were washed with 50ml HB after use and the wash was collected and added. The homogenate was then centrifuged at 14,000g for 20 minutes at 4M-0C in order to pellet the cell fraction containing SN (Quinlan, Philpot et al. 1999, Raab-Graham, Haddick et al. 2006, Sosanya, Huang et al. 2013). The supernatant was removed and the pellet snap frozen and stored at -80M-0C for SN RNA analysis. Adult (two month old) C57BL/6J female mice were maintained at the University of Texas at Austin Animal Research Center. Mice were given a one-week acclimation period in combined housing and another week to acclimate to the bottle position in individual housing. Food and water were provided ad libitum and monitored daily, as were the temperature and light/dark cycles. We extracted total RNA from the extended amygdala of 8 alcohol treated mice and 13 controls and 42 microarrays were used for the alcohol-control analysis, 21 SN and 21 paired TH. Total RNA was extracted from the SN and TH with the Direct-Zol RNA extraction kit (Zymo, Japan), using the small IC extraction columns according to manufacturerM-bM-^@M-^Ys instructions. The RNA was quantified using nanodrop (NanoDrop 1000, Thermo Fisher Scientific Inc., IL) and assayed for quality using Agilent 2100 Tape Station (Agilent Technologies, CA). The cut-off criteria were set on 280/260 > 1.7, RIN > 6.5, and amount of total RNA > 500ng. We used two validation methods for the preparation: flow cytometer analysis of SN filtration according to (Gylys, Fein et al. 2004, Sokolow, Henkins et al. 2012) and immunocytochemistry (Gylys, Fein et al. 2004, Williams, Mehrian Shai et al. 2009). According to manufacturerM-bM-^@M-^Ys instructions. Protocol Type labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Comment[SecondaryAccession] GSE51730 Comment[GEOReleaseDate] 2013-10-28 Comment[ArrayExpressSubmissionDate] 2013-10-25 Comment[GEOLastUpdateDate] 2013-10-30 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE51730_Detected_Control_SN_vs_TH.txt Comment[AdditionalFile:Data2] GSE51730_Detected_SN_control_vs_alcohol.txt Comment[AdditionalFile:Data3] GSE51730_Detected_TH_control_vs_alcohol.txt Comment[AdditionalFile:Data4] GSE51730_Norm._Control_SN_vs_TH.txt Comment[AdditionalFile:Data5] GSE51730_Norm._SN_control_vs_alcohol.txt Comment[AdditionalFile:Data6] GSE51730_Norm._TH_control_vs_alcohol.txt SDRF File E-GEOD-51730.sdrf.txt