A conditional, tissue specific 5-HT1B knockout mouse

描述(申请人提供)：5-HT1B受体在大脑不同类型的神经元中表达，在那里它们作为突触前终末的抑制性受体。当由5-羟色胺能神经元表达时，它们是自体受体，而在其他类型的神经元中，它们作为异源受体。5-HT1B自体受体和异源受体的作用一直很难区分，因为它们在大多数大脑区域混合在一起，尽管它们调节着不同神经递质的释放。此外，5-HT1B受体的结构性敲除具有复杂的表型，可能主要反映发育代偿，而不是告知这些受体在成人大脑功能中的作用。因此，需要两种类型的条件表达作为工具来研究不同神经元中5-HT1B受体在复杂情绪行为中的作用：基因敲除的时间和表型特异性。这项建议旨在通过创造一种新的转基因小鼠来解决这个问题，这种转基因小鼠将允许使用现有的CRE和FLP驱动系有条件和特定细胞类型的删除或保护5-HT1B受体的表达。拟议中的转基因小鼠具有创新性，原因有几个。1.有条件的、特定细胞类型的Cre表达将在靶神经元中删除该基因。2.有条件的、细胞类型特异性的FLP表达将切除一个loxP位点，从而选择性地保护这些神经元中的5-HT1B基因，而其他地方的5-HT1B受体随后可以被CRE敲除。3.靶向构建体旨在最大限度地减少5-HT1B表达在缺失前的基线降低的机会(即亚型)。4.这一策略可以应用于许多不同的情况，利用任何可用的CRE和FLP驱动系靶向(或保护)5-HT1B受体。对于这个修订的R21建议，我们将把我们的特征集中在5-HT1B自身受体的条件性敲除上，只在5-羟色胺能神经元中。在目标1中，我们将构建靶向结构，通过同源重组表达，获得转基因小鼠，并对小鼠的行为表型进行鉴定。在目标2中，我们将研究选择性敲除5-HT1B自身受体(在5-羟色胺神经元中)对5-羟色胺转运体功能和条件性恐惧的影响，这是一种与许多精神疾病相关的动物模型。这将使我们能够明确地探讨我们的假设，即5-HT1B自身受体是5-HT1B介导的恐惧行为减少的主要部位。在未来，这将是可能的 通过使用广泛的其他CRE和FLP驱动系来检查5-HT1B药物的作用部位，以研究其他重要的神经生物学问题，包括药物成瘾模型、饮食行为调节和呼吸控制在婴儿猝死综合征动物模型中的作用。

DESCRIPTION (provided by applicant): 5-HT1B receptors are expressed in diverse neuron types throughout the brain where they act as inhibitory receptors on presynaptic terminals. When expressed by serotonergic neurons they are autoreceptors whereas in other types of neurons they act as heteroreceptors. It has been difficult to sort out the role of 5-HT1B autoreceptors and heteroreceptors because they are intermixed in most brain regions, even though they are regulating the release of different neurotransmitters. Furthermore, constitutive knockout of 5-HT1B receptors have a complex phenotype that may reflect developmental compensations predominantly, instead of informing about the role of these receptors in adult brain function. Therefore, two types of conditional expression are needed as tools to investigate the contribution of 5-HT1B receptors in different neurons to complex emotional behavior: temporal and phenotype specificity of gene knockout. This proposal intends to solve this problem by creating a new transgenic mouse that will allow conditional and cell type-specific deletion or protection of 5-HT1B receptor expression using available Cre and Flp driver lines. The proposed transgenic mouse is innovative for several reasons. 1. Conditional, cell type-specific expression of Cre will delete the gene in the targeted neurons. 2. Conditional, cell type-specific expression of Flp will excise a loxP site thereby selectively protecting the 5-HT1B gene in those neurons while remaining 5-HT1B receptors elsewhere can be subsequently knocked out by Cre. 3. The targeting construct is designed to minimize the chances of baseline reduction in 5-HT1B expression prior to deletion (i.e. hypomorphism). 4. This strategy can be applied to many different situations to target (or preserve) 5-HT1B receptors with any available Cre and Flp driver lines. For this revised R21 proposal we will focus our characterization on the conditional knockout of 5-HT1B autoreceptors just in serotonergic neurons. In Aim 1 we will construct the targeting construct, express it via homologous recombination, derive transgenic mice, and characterize the behavioral phenotype of the mice. In Aim 2 we will examine the impact of the selective knockout of 5-HT1B autoreceptors (in serotonin neurons) on serotonin transporter function and conditioned fear, an animal model relevant to a number of psychiatric disorders. This will allow us to probe our hypothesis that 5-HT1B autoreceptors are the primary site of 5-HT1B-mediated reductions in fear behavior definitively. In the future it will be possible to examine the site of action of 5-HT1B drugs by using a wide range of other Cre and Flp driver lines to investigate other important neurobiological problems including models of drug addiction, regulation of eating behaviors, and control of respiration in animal models of Sudden Infant Death Syndrome.