Endogenous neurosteroids constrain network activity and limit seizure susceptibility

项目摘要 有广泛的临床前证据表明，神经活性类固醇具有抗惊厥作用，人们认为 通过它们作为GABAA受体上的正变构调节器(PAM)的作用而被介导 (GABAAR)。这些研究依赖于外源性神经活性类固醇的管理，尽管， 神经类固醇也可以在大脑中合成。然而，我们对它的作用知之甚少。 内源性神经类固醇由于缺乏必要的工具来研究其功能。限速 参与神经类固醇合成的酶，5α还原酶1或2，在海马区表达并给予 他们作为GABAAR的PAM，非常适合限制该地区的网络兴奋性。至 为了研究内源性神经类固醇的功能，我们建立了新的小鼠模型(Floxed Srd5a1- IRES-GFP和Floted Srd5a2-IRES-tdTomato)实现了可视化和量化 这些酶的表达以及在一种细胞类型中敲除这些酶的能力-以及 大脑区域特有的方式。在这里，我们建议使用这些新的工具来检验以下假设 内源性神经类固醇抑制神经元兴奋性及5-α-还原酶1和2表达缺陷 在海马体中对癫痫易感性有贡献。我们将确定5α还原酶1和/或2 慢性癫痫小鼠(特异性靶点1)的表达发生改变，如果5α还原酶1和/或2丢失 海马区表达增加网络兴奋性和急性癫痫敏感性(特定目标2) 或增加慢性癫痫小鼠的癫痫发作频率(具体目标3)。

Project Summary There is extensive preclinical evidence for anticonvulsant effects of neuroactive steroids, which are thought to be mediated through their actions as positive allosteric modulators (PAMs) on GABAA receptors (GABAARs). These studies have relied on the administration of exogenous neuroactive steroids, although, neurosteroids can also be synthesized in the brain. However, we know very little about the function of endogenous neurosteroids due to the lack of tools necessary to investigate their function. The rate-limiting enzymes involved in neurosteroid synthesis, 5α-reductase 1 or 2, are expressed in the hippocampus and given their actions as PAMs at GABAARs, they are well-suited to constrain network excitability in this region. To investigate the function of endogenous neurosteroids, we generated novel mouse models (floxed Srd5a1- IRES-GFP and floxed Srd5a2-IRES-tdTomato) enabling visualization and quantification of changes in the expression of these enzymes associated as well as the ability to knockout these enzymes in a cell type- and brain region-specific manner. Here we propose to utilize these novel tools to test the hypothesis that endogenous neurosteroids constrain neuronal excitability and that deficits in 5α-reductase 1 and 2 expression in the hippocampus contributes to seizure susceptibility. We will determine whether 5α-reductase 1 and/or 2 expression is altered in chronically epileptic mice (Specific Aim 1) and if loss of 5α-reductase 1 and/or 2 expression in the hippocampus increases network excitability and acute seizure susceptibility (Specific Aim 2) or increases seizure frequency in chronically epileptic mice (Specific Aim 3).