Project description:Catechol-O-methyltransferase (COMT) is an ubiquitously expressed enzyme that maintains basic biologic functions by inactivating catechol substrates. In humans, polymorphic variance at the COMT locus has been associated with modulation of pain sensitivity (Andersen & Skorpen, 2009) and risk for developing psychiatric disorders (Harrison & Tunbridge, 2008). A functional haplotype associated with increased pain sensitivity was shown to result in decreased COMT activity by altering mRNA secondary structure-dependent protein translation (Nackley et al., 2006). However, the exact mechanisms whereby COMT modulates pain sensitivity and behavior remain unclear and can be further studied in animal models. We have pursued a genome-wide approach to examining gene expression in multiple brain regions in inbred strains of mice and have discovered that Comt1 is differentially expressed. This expression difference was validated with qPCR. A B2-B4 Short Interspersed Element (SINE) was inserted in the 3'UTR of Comt1 in 14 strains that also shared a common haplotype. Experiments using mammalian expression vectors of full-length cDNA clones with and without the SINE element demonstrate that strains with the SINE haplotype (+SINE) have greater Comt1 enzymatic activity. +SINE mice also exhibit behavioral differences in anxiety assays and decreased pain sensitivity. These results suggest that a haplotype, defined by a 3'UTR B2-B4 SINE element, regulates Comt1 expression and mouse behavior. Brain region samples were isolated from male and female mice of 29 strains (n = 3), 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, BTBR T+ tf/J, BUB/BnJ , C3H/HeJ, C57BL/6J, C57BR/cdJ, C58/J, CBA/J, CE/J, DBA/2J, FVB/NJ, I/LnJ, KK/HIJ, MA/MyJ, MRL/MpJ, NOD/LtJ, NON/LtJ, NZO/HILTJ, NZW/LacJ, P/J, PL/J, RIIIS/J, SJL/J, SM/J, SWR/J, and WSB/EiJ. Eight to ten week old male and female mice of each strain were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were habituated for one week prior to tissue collection. Mice were sacrificed by cervical dislocation without anesthesia to avoid gene expression differences in response to anesthetic. All dissections were conducted between the hours of 9:00AM to 11:30AM. Prefrontal cortex and nucleus accumbens were dissected as follows. Immediately following euthanasia, mice were decapitated and the whole brain was removed from the skull. The brain was then transferred, ventral side up, to an ice-cold brain matrix with 0.5mm spacing (505C Braintree Scientific). A single razor blade was placed into the first space on the brain matrix and the rostral surface of the brain was placed in the matrix and touching this blade. Thin, double-edged razor blades were placed in the twelve most anterior spaces. Following removal from the matrix, the 0.5 mm brain slices were placed flat onto an ice-cold dissection stage and specific regions dissected using anatomical landmarks as described below. The prefrontal cortex was taken from the slice corresponding to approximately 2.5 mm to 2.0 mm anterior to Bregma. To do so, a V-shaped wedge was made just medial to the corpus callosum with the apex terminating at about the lateral ventricle (viewed from the caudal side of the slice). Nucleus accumbens was taken from the adjacent slice approximately 2.0mm to 1.5mm anterior to Bregma. To isolate nucleus accumbens, 1mm punches were taken just ventromedial to the anterior commissure. All of the array data in this Series are from male mice.
2010-02-23 | E-GEOD-20160 | biostudies-arrayexpress
