STRESS ALLOSTASIS: CRF, SEROTONIN AND THE BNST

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在报告期间，我们使用单细胞RT-PCR来补充我们对BNST中5-羟色胺受体功能的生理学研究。我们现在的数据表明，单个BNST神经元可以表达多种5-羟色胺受体，慢性束缚应激可以显著改变这些神经元中特定的5-羟色胺受体mRNA的表达模式。在一定程度上，应激反应可以通过重复注射皮质醇来模拟，这表明这种变化可能是在面对长期应激时调节BNST神经元兴奋性的反馈反应的一部分。值得注意的是，我们也有蛋白质印迹和行为数据表明，mRNA的变化在BNST中有相应的肽表达变化，并且特定肽(Kv4.2)的丢失与慢性束缚应激动物焦虑样行为的增加呈正相关。我们还继续我们的单细胞RT-PCR检测特定的离子通道亚单位mRNA的表达，以确定哪些离子通道亚单位对BNST中不同类型的细胞(I-III型)表达的固有膜电流起作用。到目前为止，我们已经确定了至少5个IA通道亚基，3个IT通道亚基和4个IH通道亚基，我们希望在2009年初提交一篇关于II型BNST神经元的分子解剖的论文。我们还开发了一种新型的转基因小鼠，其中CRF启动子用于驱动绿色荧光蛋白(GFP)的表达。我们已经证明，GFP的表达仅限于那些被预测包含大量CRF神经元的区域，包括BNST。对含有CRF的神经元特性的初步生理学特征表明，它们对应于在大鼠组织中看到的BNST神经元的III型。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the reporting period we used single cell RT-PCR to complement our physiological study of 5HT receptor function in the BNST. We now have data suggesting that individual BNST neurons can express multiple 5HT receptors, and that chronic restraint stress can dramatically alter the expression patterns of specific 5HT receptor mRNA in these same neurons. In part, the stress response can be mimicked by repeated cortisol injections suggesting that the alteration may be part of a feedback response to regulate BNST neuronal excitability in the face of prolonged stress. Significantly, we also have Western blot- and behavioral data to show that the mRNA change has a corresponding change in peptide expression in the BNST and that loss of a particular peptide (Kv4.2) is positively correlated with an increase in anxiety-like behavior in chronic restraint stress animals. We also continued our single cell RT-PCR examination of specific ion channel subunit mRNA expression in order to determine which ion channel subunits contribute to the intrinsic membrane currents that are expressed by distinct cell types in the BNST (Types I - III). To date we have characterized at least 5 IA channel subunits, 3 IT channel subunits and 4 IH channel subunits, and we hope to submit a paper on the molecular dissection of Type II BNST neurons early in 2009. We have also developed a novel transgenic mouse in which the CRF promoter is used to drive the expression of green fluorescent protein (GFP). We have shown that GFP expression is restricted to those areas predicted to contain high numbers of CRF neurons, including the BNST. Preliminary physiological characterization of the properties of the CRF-containing neurons suggests that they correspond to the Type III subtype of BNST neuron seen in rat tissue.