VCU BXD PFC CIE Air Masked ComBat M430 2.0 (Apr16) RMA **

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In order to elucidate the molecular mechanisms underlying individual variation in sensitivity to ethanol we profiled the prefrontal cortex transcriptomes of two inbred strains that exhibit divergent responses to acute ethanol, the C57BL6/J (B6) and DBA/2J (D2) strains, as well as 27 members of the BXD recombinant inbred panel, which was derived from a B6 x D2 cross. With this dataset we were able to identify several gene co-expression networks that were robustly altered by acute ethanol across the BXD panel. These ethanol-responsive gene-enriched networks were heavily populated by genes regulating synaptic transmission and neuroplasticity, and showed strong genetic linkage to discreet chromosomal loci. Network-based measurements of node importance identified several hub genes as established regulators of ethanol response phenotypes, while other hubs represent novel candidate modulators of ethanol responses.

Experiment design

This data set was generated concurrently with the VCU saline prefrontal cortex BXD RMA data and therefore consisted of 64 microarrays processed in 5 groups of 8 to 16 microarrays during the month of September 2006. All RNA extractions, cRNA synthesis, and hybridizations were randomized across strain and treatment groups to minimize batch effects.

Many of the techniques used to generate this data set are described in a recent publication in the Journal of Neuroscience.

Animals were injected intraperitoneally (IP) with saline or 1.8 g/kg of ethanol. As part of a parallel study of ethanol induced anxiolysis, all mice underwent behavioral testing that included 15 minutes of restraint in a 50 mL conical tube followed by 10 minutes in a light-dark chamber. Mice were killed by cervical dislocation four hours following IP injection. Immediately thereafter, brains were extracted and chilled for 1 minute in iced phosphate buffer before being microdissected into 8 constituent regions, including the medial prefrontal cortex. Samples were randomly assigned to batch groups prior to total RNA extraction, cRNA synthesis and hybridization. Each microarray represent a pooling of 4-5 animals.

About cases

This BXD data set provides estimates of mRNA expression in the prefrontal cortex following ethanol treatment across 27 BXD recombinant inbred strains and their B6 and D2 progenitor strains. All samples are from a total of 468 adult male animals obtained from Jackson Laboratory and raised in a standard laboratory environment. An average of 8 males per strain was used to measure anxiety-like behavior in response to restraint and treatment with 1.8g/kg ethanol in the light-dark transition model of anxiety. Four hours after treatment, animals were rapidly sacrificed by cervical dislocation, brains were removed, cooled and microdissected as previously described (Kerns et al., J. Neurosci. 25:2255, 2005). All RNA isolation and subsequent probe generation and hybridization to microarrays were completed using a supervised randomization procedure to minimize batch effects. Affymetrix M430 type 2.0 microarrays were used for hybridization using standard procedures. Expression analysis was conducted by estimating the relative abundance of over 45,000 transcripts in the prefrontal cortex following ethanol treatment using the Robust Multichip Average (RMA) method.

About tissue

All animals were obtained at 8-9 weeks of age from the Jackson Laboratory (Bar Harbor, ME) and were treated, behaviorally tested and brains dissected by Alex Putman and colleagues at VCU. Following an hour acclimation period to the behavioral room, animals were restrained for 15 minutes, immediately injected (I.P.) with either saline (0.9%) or 1.8g/kg ethanol, and 5 minutes later placed in the light-dark box for a 10-minute test session. All behavioral testing occurred between 10 AM and 1 PM during the light phase over a 12 month period beginning August 2005. Four hours after treatment, animals were rapidly sacrificed by cervical dislocation, brains were removed, cooled and microdissected. Prefrontal cortex tissue was isolated by microdissection using a wedge-shaped slice taken from a 4 mm thick brain slice extending rostrally from the optic chiasm. The wedge was centered on the inter-hemispheric fissure and extending 2 mm laterally on each side and ventrally to just above the corpus callosum. This tissue and all other brain regions were dissected in less than 5 minutes per mouse and were immediately frozen in liquid nitrogen followed by storage at -80 oC prior to RNA isolation. A pool of dissected tissue from 3 mice of the same strain was used to generate RNA samples. All RNA samples were extracted at VCU by Alex Putman during October 2006 and the order of RNA isolation was randomized across all strains and treatment groups (since saline treated animals were processed concurrently).

GEO Accession: GSE28515

About platform

[Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array


Miles MF, Putman AH, Vorster PJ, Wolen AR


Data were generated with funds to Michael F Miles from the NIAAA.