Cell type specific gene expression in the visual cortex

描述（由申请人提供）：尽管有一百年的解剖学研究和近五十年的电生理学研究，但对构成视觉皮层的神经元细胞类型缺乏充分的了解。关于基于形态学、轴突投射、细胞生理学和神经化学定义的细胞类型如何相互关联或与由其体内反应特性定义的神经元功能类别相关，存在广泛的分歧。尽管这个问题很困难，但如果在啮齿动物中进行的细胞和分子工作与食肉动物和灵长类动物中进行的体内生理学以及人类的临床观察有关，那么进展是至关重要的。在这里，我们建议结合联合收割机解剖学，生理学和基因表达分析，发现一套标记，可用于识别皮质细胞类别。在转基因小鼠中荧光蛋白表达的基础上，已经确定了共享层状和亚层状位置、树突形态和轴突靶点的锥体神经元的亚群。将使用荧光激活细胞分选来分离这些神经元，并且将使用Affyssin基因芯片来测量它们的基因表达模式。将使用多种小鼠品系，其中变体荧光蛋白在不同的神经元亚群中表达，以允许比较不同细胞类型的基因表达。切片中的全细胞记录将用于表征每个候选神经元细胞类别的内在电生理学。它们的形态学特性将从细胞内填充中测量;它们的突触连接将从双全细胞记录中评估，并且它们的疑似长距离投射将使用逆行示踪剂来确认。这项工作可以作为跨区域，跨物种和跨发展确定同源皮质细胞类型的基础。此外，在确定的神经元类型的基因表达的测定可以提供一个更有选择性和灵敏度的基因表达的变化，由改变的感觉经验或神经疾病诱导的测定。

DESCRIPTION (provided by applicant): Despite a hundred years of anatomical work and nearly fifty years of electrophysiology, a full understanding of the neuronal cell types that comprise the visual cortex is lacking. There is widespread disagreement as to how cell types defined on the basis of morphology, axonal projections, cellular physiology, and neurochemistry relate to one another or to functional categories of neurons defined by their response properties in vivo. Despite the difficulty of the problem, progress is crucial if cellular and molecular work done in rodents is to be related to in vivo physiology performed in carnivores and primates, and to clinical observations in humans. Here we propose to combine anatomy, physiology and gene expression analysis to discover sets of markers that can be used to identify cortical cell classes. Subsets of pyramidal neurons that share laminar and sublaminar position, dendritic morphology and axonal targets have been identified on the basis of expression of fluorescent proteins in transgenic mice. Fluorescence activated cell sorting will be used to isolate these neurons and Affymetrix gene chips will be used to measure their patterns of gene expression. Multiple strains of mice will be used in which variant fluorescent proteins are expressed in different subsets of neurons to allow comparisons of gene expression across different cell types. Whole cell recording in slices will be used to characterize the intrinsic electrophysiology of each candidate neuronal cell class. Their morphological properties will be measured from intracellular fills; their synaptic connectivity will be assessed from dual whole cell recording, and their suspected long range projections will be confirmed using retrograde tracers. This work may serve as a foundation for identifying homologous cortical cell types across regions, across species and across development. In addition, the assays of gene expression in identified neuronal types may provide a more selective and sensitive assay of changes in gene expression induced by altered sensory experience or by neurological diseases.