A novel genetic and electrophysiological approach to study hippocampal neurons

描述（由申请人提供）：海马体是边缘系统的一部分，负责调节学习和记忆，并与癫痫有关。我们正在开发一种多学科的方法来研究海马中兴奋性和抑制性神经元的发育和功能。利用电穿孔技术在子宫内转染海马干细胞，我们将引入荧光蛋白标记物以及TrkB酪氨酸激酶受体的突变版本到海马回路的特定亚区。这些外源基因的表达将被暂时控制，通过将它们置于可诱导基因启动子的控制之下，使它们的表达与神经元发育的特定阶段一致。这些分子变化的功能影响将通过使用免疫化学染色表征细胞分化和使用膜片钳记录的直接电生理测量来评估。这些实验将定义一种新的、快速的方法，通过引入生发细胞中的分子变化来评估海马神经元特定亚群的发育、分配和突触生理学。这种方法将导致海马功能研究的技术进步，并将有助于了解癫痫和智力迟钝等疾病中异常突触传递的潜在原因。公共卫生相关性：在这个项目中，我们寻求开发跟踪海马体神经元发育和改变基因表达的方法，以实现快速和具有成本效益的功能测试。特别是，我们将评估TrkB神经营养因子受体在海马神经元迁移和突触可塑性中的作用。为了研究TrkB扰动的产前和产后影响，我们设计了可诱导质粒结构，我们可以在给予他莫昔芬后启动转基因的表达。这项工作将提高我们阐明正常发育机制的能力，并允许对癫痫和智力迟钝的原因进行快速检测。

DESCRIPTION (provided by applicant): The hippocampus, part of the limbic system, mediates learning and memory and is implicated in epilepsy. We are developing a multidisciplinary approach to study development and function of the excitatory and inhibitory neurons in the hippocampus. Using electroporation to transfect the hippocampal stem cells in utero, we will introduce fluorescent protein markers as well as mutated versions of the TrkB tyrosine kinase receptor into specific subregions of the hippocampal circuitry. The expression of these exogenous genes will be controlled temporally by placing them under the control of inducible gene promoters so that their expression coincides with particular phases of neuronal development. The functional effects of these molecular changes will be assessed by characterizing cell differentiation using immunochemical staining and by direct electrophysiogical measurements using patch clamp recording. These experiments will define a novel and rapid method for assessing the development, allocation and synaptic physiology of specific subpopulations of hippocampal neurons by introducing molecular changes in their germinal cells. This method will result in a technical advance for the study of hippocampal function and will be useful for understanding the underlying causes of abnormal synaptic transmission in diseases including epilepsy and mental retardation. PUBLIC HEALTH RELEVANCE:In this project we seek to develop methods for tracking the development and changing the gene expression of neurons in the hippocampus to enable rapid and cost-effective functional tests. In particular we will be assessing the role of the TrkB neurotrophin receptor in hippocampal neuronal migration and synaptic plasticity. To study both prenatal and postnatal effects of TrkB perturbation, we have engineered inducible plasmid constructs with which we can initiate expression of the transgene upon administration of tamoxifen. This work will enhance our ability to elucidate normal developmental mechanisms and allow rapid testing on causes of epilepsy and mental retardation.