Gene Expression Data from Prefrontal Cortex and Nucleus Accumbens from Inbred Strains of Mice

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.邻苯二酚-O-甲基转移酶（Catechol-O-methyltransferase, COMT）是一种广泛表达的酶，通过灭活儿茶酚底物维持机体基础生物学功能。在人类中，COMT基因座的多态性变异与疼痛敏感性调节（Andersen & Skorpen, 2009）及精神疾病发病风险相关（Harrison & Tunbridge, 2008）。既往研究显示，一种与疼痛敏感性升高相关的功能性单倍型，可通过改变mRNA二级结构依赖的蛋白质翻译过程降低COMT活性（Nackley et al., 2006）。然而，COMT调节疼痛敏感性与行为的确切机制仍不明确，可通过动物模型开展进一步研究。 本研究采用全基因组分析方法，对近交系小鼠的多个脑区基因表达进行检测，发现Comt1存在差异表达。该表达差异已通过定量聚合酶链式反应（quantitative polymerase chain reaction, qPCR）验证。在14个共享同一单倍型的小鼠品系中，Comt1的3'非翻译区（3'UTR）插入了一段B2-B4短散在重复元件（Short Interspersed Element, SINE）。 利用携带或不携带该SINE元件的全长cDNA克隆构建的哺乳动物表达载体进行实验，结果显示带有SINE单倍型（+SINE）的品系其Comt1酶活性更高。+SINE小鼠在焦虑相关行为学实验中表现出行为差异，且疼痛敏感性降低。上述结果提示，由3'UTR区域的B2-B4 SINE元件定义的单倍型可调节Comt1的表达及小鼠行为表型。 本研究采集了29个品系的雌雄小鼠的脑区样本，每个品系的样本量为n=3，涉及品系包括：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及WSB/EiJ。 各品系8~10周龄的雌雄小鼠均购自美国缅因州巴尔港的杰克逊实验室（Jackson Laboratory）。小鼠在组织采集前适应性饲养1周。为避免麻醉剂诱导的基因表达差异，本研究采用颈椎脱臼法处死小鼠，未使用麻醉剂。所有解剖操作均在上午9:00至11:30之间进行。 前额叶皮层与伏隔核的解剖分离方法如下：小鼠处死后立即断头，将全脑从颅骨中取出，腹侧朝上放置于间距为0.5mm的冰浴脑切片模具（505C, Braintree Scientific公司）。将单面刀片置于模具的第一个卡槽内，将脑的吻侧端贴合该刀片放置。随后将双面薄刀片嵌入最前方的12个卡槽内。从模具中取出0.5mm厚的脑片后，将其平铺于冰冷却的解剖台，通过解剖学标记分离特定脑区，具体步骤为：前额叶皮层取自相对于前囟（Bregma）前侧约2.5mm至2.0mm的脑片，操作时在胼胝体内侧制作V形楔形区域，其顶点约终止于侧脑室（从脑片尾侧观察）；伏隔核取自相对于前囟前侧约2.0mm至1.5mm的相邻脑片，通过在前连合腹内侧进行1mm直径的组织穿刺取样分离。本研究的所有基因芯片数据均来自雄性小鼠。