Cell type specific gene expression in the visual cortex

描述(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，但对构成视觉皮质的神经细胞类型缺乏充分的了解。对于基于形态、轴突投射、细胞生理学和神经化学的细胞类型如何相互联系，或者与由其在体内的反应特性定义的神经元的功能类别之间的关系，存在着广泛的分歧。尽管这个问题很困难，但如果要将啮齿动物的细胞和分子工作与肉食动物和灵长类动物的体内生理学以及人类的临床观察联系起来，进展是至关重要的。在这里，我们建议结合解剖学、生理学和基因表达分析来发现可用于识别皮质细胞类别的标记集。在转基因小鼠的荧光蛋白表达的基础上，已经鉴定出具有相同的板层和亚板下位置、树突形态和轴突靶标的锥体神经元亚群。将使用荧光激活的细胞分选来分离这些神经元，并使用Affymetrix基因芯片来测量它们的基因表达模式。将使用多个品系的小鼠，其中不同的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。切片中的整个细胞记录将被用来表征每个候选神经细胞类别的内在电生理学。它们的形态特征将通过细胞内填充来测量；它们的突触连通性将通过双重全细胞记录来评估，其可疑的长距离投射将使用逆行示踪剂进行确认。这项工作可以作为识别跨地区、跨物种和跨发育的同源皮质细胞类型的基础。此外，已确定的神经元类型的基因表达分析可能提供更具选择性和敏感性的基因表达变化分析，这些变化是由感觉经验改变或神经疾病引起的。

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.