SERT KO rats are a model of sex specific visceral pain

描述(申请人提供)：这个项目将测试5-羟色胺(5-羟色胺，5-羟色胺)和雌激素信号相互作用以增加供应结肠的初级传入神经元的兴奋性的假设。兴奋性的增加是由于离子通道表达的变化导致雌性大鼠的内脏高敏感性。这一假设将是 用雄性和雌性5-羟色胺转运体(SERT)基因敲除(KO)大鼠进行测试，我们认为这是一种独特的性别特异性内脏高敏感性的动物模型。内脏疼痛的潜在病理生理学机制尚不清楚，部分原因是缺乏可以进行机械性和干预性研究的动物模型。然而，已发表的数据表明，5-羟色胺信号的改变可能在人类中发挥作用。此外，内脏痛在女性中比在男性中更常见，这表明5-羟色胺与性别在内脏痛的发生中存在交互作用。总体假设将在3个具体目标上进行检验。具体目的1将验证这样一种假设，即野生型(WT)和SERT KO大鼠的结肠细胞外5-羟色胺的利用度增加，而肠嗜铬细胞(EC)的5-羟色胺释放不受SERT KO的影响。安培法将测量黏膜附近的5-羟色胺对机械和化学粘膜刺激的反应。免疫组织化学(IHC)和Western印迹技术将用于验证SERT缺失，并评估WT和SERT KO大鼠肠道中含有5-羟色胺的EC细胞。将评估多巴胺和去甲肾上腺素转运体以及有机阳离子转运体的IHC定位，以确定它们在SERT KO大鼠中的表达是否增加。特定目标2将验证雌激素与5-羟色胺相互作用导致雌性SERT KO大鼠内脏过敏的假设。我们将利用内脏运动对结直肠球囊扩张的反应来测量完整的和去卵巢的雌性WT和SERT KO大鼠在接受和不接受雌激素替代后的内脏敏感性。在具体目标3中，将研究短期原代培养中保持的结肠投射感觉神经元的功能特性。这些研究将验证这样一种假设，即当使用全细胞膜片钳方法在体外研究SERT KO大鼠的结肠投射感觉神经时，雌性SERT KO大鼠的兴奋性增加。兴奋性的增加被认为是由于5-羟色胺和雌激素在感觉神经元上的相互作用，导致了河豚毒素抗性钠通道的上调。这些研究将表明，在雌性SERT KO大鼠中增加5-羟色胺的可获得性改变内脏敏感性，就像在女性人类肠易激综合征患者中发生的那样。这些数据表明，SERT KO大鼠是研究肠道感觉神经供应变化导致内脏过敏的模型。

DESCRIPTION (provided by applicant): This project will test the hypothesis that 5-hydroxytryptamine (5-HT, serotonin) and estrogen signaling interact to increase excitability of primary afferent neurons supplying the colon. Increased excitability is caused by changes in ion channel expression that result in visceral hypersensitivity in female rats. This hypothesis will be tested using male and female serotonin transporter (SERT) knockout (KO) rats, which we propose is a unique animal model of gender specific visceral hypersensitivity. The underlying pathophysiology of visceral pain is unclear and this is partly due to a lack of animal models where mechanistic and interventional studies can be conducted. However, published data indicate that alterations in 5-HT signaling may play a role in humans. In addition, visceral pain i more common in women than in men, suggesting that there are interactions between 5-HT and gender in the genesis of visceral pain. The overall hypothesis will be tested in 3 specific aims. Specific aim 1 will test the hypothesis that there is increased extracellular availability of 5-HT n vitro in the colon of wild type (WT) and SERT KO rats and that 5-HT release from enterochromaffin (EC) cells is unaffected by the SERT KO. Amperometry will measure 5-HT near the mucosa in response to mechanical and chemical mucosal stimulation. Immunohistochemical (IHC) and Western blot techniques will be used to verify SERT deletion and to assess 5-HT-containing EC cells in the gut of WT and SERT KO rats. IHC localization of the dopamine and norepinephrine transporters and organic cation transporters will be assessed to determine if their expression increases in SERT KO rats. Specific aim 2 will test the hypothesis that estrogen interacts with 5-HT to cause visceral hypersensitivity in female SERT KO rats. The visceromotor response to colorectal balloon distention will be used to measure visceral sensitivity in intact and ovariectomized female WT and SERT KO rats with and without estrogen replacement. In Specific Aim 3, the functional properties of colon projecting sensory neurons maintained in short term primary culture will be studied. These studies will test the hypothesis that colon projecting sensory nerves from female but not male SERT KO rats exhibit increased excitability when studied using whole cell patch clamp methods in vitro. The increased excitability is proposed to be due to interactions between 5-HT and estrogen on sensory neurons that lead to upregulation of tetrodotoxin-resistant sodium channels. These studies will show that increased 5-HT availability in female SERT KO rats alters visceral sensitivity as occurs in female human irritable bowel syndrome patients. The data would indicate that the SERT KO rat is a model for studying changes in the sensory nerve supply of the gut that leads to visceral hypersensitivity.