Analysis of Area-Specific Expression Patterns of RORbeta, ER81 and Nurr1 mRNAs in Rat Neocortex by Double In Situ Hybridization and Cortical Box Method Junya Hirokawa1,2., Akiya Watakabe1,2., Sonoko Ohsawa1, Tetsuo Yamamori1,2* 1 Division of Brain Biology, National Institute for Basic Biology, Okazaki, Japan, 2 Department of Basic Biology, The Graduate University for Advanced Studies, Okazaki, Japan Abstract Background: The mammalian neocortex is subdivided into many areas, each of which exhibits distinctive lamina architecture. To investigate such area differences in detail, we chose three genes for comparative analyses, namely, RORbeta, ER81 and Nurr1, mRNAs of which have been reported to be mainly expressed in layers 4, 5 and 6, respectively. To analyze their qualitative and quantitative coexpression profiles in the rat neocortex, we used double in situ hybridization (ISH) histochemistry and cortical box method which we previously developed to integrate the data of different staining and individuals in a standard three-dimensional space. Principal Findings: Our new approach resulted in three main observations. First, the three genes showed unique area distribution patterns that are mostly complementary to one another. The patterns revealed by cortical box method matched well with the cytoarchitectonic areas defined by Nissl staining. Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized. Third, principal component analysis showed that the order of hierarchical processing in the cortex correlates well with the expression profiles of these three genes. Based on this analysis, the dysgranular zone (DZ) in the somatosensory area was considered to exhibit a profile of a higher order area, which is consistent with previous proposal. Conclusions/Significance: The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex. In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex. Citation: Hirokawa J, Watakabe A, Ohsawa S, Yamamori T (2008) Analysis of Area-Specific Expression Patterns of RORbeta, ER81 and Nurr1 mRNAs in Rat Neocortex by Double In Situ Hybridization and Cortical Box Method. PLoS ONE 3(9): e3266. doi:10.1371/journal.pone.0003266 Editor: Olaf Sporns, Indiana University, United States of America Received June 30, 2008; Accepted September 4, 2008; Published September 25, 2008 Copyright: 2008 Hirokawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Grant-in-Aid for Scientific Research (KAKENHI) 17024055 (to T.Y.) and 19500304 (to A. W.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yamamori@nibb.ac.jp . These authors contributed equally to this work. Introduction The mammalian neocortex consists of many areas that are defined on the basis of unique connectional and functional properties [1], [2]. In accordance with functional specialization, these areas exhibit various differences in terms of their structural configurations as revealed by Nissl staining and other conventional histological techniques [1], [3]. More recently, it has become possible to selectively visualize particular neocortical structures by techniques to map gene products, such as immunocytochemistry [4], [5], receptor autoradiography [6–9], and in situ hybridization histochemistry (ISH) [10–12]. For example, several genes have been shown to exhibit layer- and area-specific expression profiles during development or in adulthood [5], [10], [11], [13–23]. For the rodent cortex, it is now possible to examine the expression data of most of the known genes in public databases [24–27]. Effective use of such information may enable us to reveal apparently hidden structures of neocortical areas, such as new sublayers and areas defined by expression of a unique set of genes. In our previous study, we have shown that layer-specific gene expressions can reveal cortical structures across areas and species [23]. Consistent with the six-layer model originally proposed by Brodmann [1], the lamina expressions of several genes were conserved across areas in monkey and mouse neocortices. At the same time, we observed various area differences in their expression patterns. For example, we observed that the width and intensity of gene expressions exhibit abrupt changes across the V1–V2 border in the monkey cortex. We also found that a subtype of excitatory neurons that express 5-HT2C receptor mRNA are localized in layer 5 in most areas, but in layer 6 in monkey V1. These observations may reflect the conspicuous difference of monkey V1 compared with other areas [28–30]. While the differences between V1 and V2 are rather conspicuous, there are often more subtle differences in other areas. These subtle differences are more PLoS ONE | www.plosone.org 1 September 2008 | Volume 3 | Issue 9 | e3266