Cell type specific gene expression in the visual cortex

描述（由申请人提供）：尽管经过了一百年的解剖工作和近五十年的电生理学，对构成视觉皮层的神经元细胞类型仍缺乏充分的了解。关于如何根据形态学、轴突投射、细胞生理学和神经化学来定义细胞类型，以及如何根据体内反应特性来定义神经元的功能类别，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研究与在食肉动物和灵长类动物中进行的体内生理学以及对人类的临床观察相关，那么取得进展是至关重要的。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层细胞类别的标记集。基于荧光蛋白在转基因小鼠中的表达，已经确定了共享层状和层下位置、树突形态和轴突靶点的锥体神经元亚群。荧光激活细胞分选将用于分离这些神经元，Affymetrix基因芯片将用于测量它们的基因表达模式。将使用多种小鼠品系，其中不同的荧光蛋白在不同的神经元亚群中表达，以便比较不同细胞类型的基因表达。全细胞切片记录将用于表征每个候选神经元细胞类别的内在电生理。它们的形态特性将从细胞内填充测量；他们的突触连通性将通过双全细胞记录进行评估，他们怀疑的远程投射将用逆行示踪剂证实。这项工作可以作为识别跨区域、跨物种和跨发育的同源皮质细胞类型的基础。此外，在已识别的神经元类型中进行基因表达的测定可以提供一种更有选择性和更敏感的测定方法，以测定由改变的感觉体验或神经系统疾病引起的基因表达变化。

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.