Figure 2. Area differences in gene expressions. The regions denoted in Fig. 1 are magnified. In these figures, the contrast was adjusted simultaneously so that the area differences can be directly compared across different areas. The layers denoted on the left side of each panel were determined in reference to the Hoechst 30442 nuclear staining. These panels are considered to correspond to cytoarchitec- tonic areas as follows: a; Oc2MM, b; Par1, c; Par2, d; Oc1, e; Te3R, f; Ect, a’; DZ, b’; Par1/Par2 border, c’; Par2, d’; Oc1, e’; Oc2L, f’; Oct. S; subiculum. Scale bar: 100 mm. doi:10.1371/journal.pone.0003266.g002 upper part of layer 5 (data not shown), despite low number of ER81-mRNA-positive cells. Such a gap does not exist in other areas (Fig. 2, panels e and f). In the laterocaudal area (e.g., Fig. 1B, Ect), neurons that expressed both ER81 and RORbeta mRNAs at moderately high level were located around the border between layers 4 and 5 (Fig. 2, panel f). These observations indicate heterogeneous lamina expression patterns of the ‘‘layer-specific’’ genes at the cellular level. The overlapping mRNA expression profiles raised the possibil- ity that the two mRNAs with different lamina specificities are coexpressed in the same cells. This was examined in high- magnification photos in Fig. 3. Although RORbeta is a marker for layer 4, it is also expressed in layer 5 throughout the rat neocortex. When we examined the coexpression of RORbeta and ER81 mRNAs by double ISH, these mRNAs were coexpressed within the same cells in layer 5 (Fig. 3A–3C). Layer 4 neurons generally expressed only RORbeta mRNA, while many layer 5 neurons expressed only ER81 mRNA. However, the layer 5 neurons expressing RORbeta mRNA mostly coexpressed ER81 mRNA. Such coexpression was observed in all the areas examined (data not shown). On the other hand, RORbeta and Nurr1 mRNAs were not coexpressed in the same neurons even in the areas with extensive intermingling (Fig. 3D–3F). Similarly, ER81 and Nurr1 mRNAs were not expressed within the same neurons (Fig. 3G–3I). This point has been shown previously for mice [23], but our data now showed the same co-expression pattern in rats. Thus, we conclude that the coexpression preferences of the three genes are common across areas, although the relative abundance and distribution are quite divergent. Cortical box method for quantitative analysis of area- specific gene expression As mentioned in the introduction, it is difficult to accurately identify cortical areas without clear-cut landmarks. To circumvent ISH Profiling of Rat Cortex Figure 3. Coexpression of RORbeta, ER81 and Nurr1 genes. (A)– (C) Double ISH of RORbeta (red) and ER81 (green) mRNAs in a somatosensory area. Note the extensive coexpression of the two genes (denoted by the arrows). (D)–(F) Double ISH of RORbeta (red) and Nurr1 (green) mRNAs in a laterocaudal area. (G)–(I) Double ISH of ER81 (red) and Nurr1 (green) mRNAs in a laterocaudal area. Note that the two genes in (D)–(F) and (G)–(I) are not coexpressed. Scale bar: 50 mm. doi:10.1371/journal.pone.0003266.g003 this problem, we applied a standardization and reconstruction procedure for the ISH samples of the serially prepared coronal sections of the posterior part of the rat cortex as follows (see also [31]). In the reconstruction, the shape of the cortex was transformed to fit into a rectangle, as illustrated in Fig. 4A. The left and right borders of the rectangle correspond to the medial ends of the cortex and the rhinal fissure, respectively, both of which can be easily determined. We also normalized the level of ISH signals so that the relative strength of the ISH signals at a given location can be compared across different data sets (Fig. 4A). Figure 4B illustrates the standardization process from the ISH data of the RORbeta gene. As shown in this figure, seventeen ISH coronal sections in total were used to cover the posterior part of one rat brain hemisphere (22.1 to 26.3 mm from Bregma) with 280 mm intervals (Fig. 4B; original images). It was already evident from the original images that there are three distinct clusters of high RORbeta signals, which roughly corresponded to the somatosensory, auditory and visual areas (delineated by yellow, red and blue lines, respectively). The middle panel of Fig. 4B shows the images transformed into seventeen rows of cortical rectangles. In these rows of images, the three clusters of high RORbeta ISH signals were now more clearly visualized (‘‘Representative’’). Importantly, once the staining intensity was standardized, we could easily integrate multiple sets of data. In the right panel of Fig. 4B, the average of six sets of samples from three PLoS ONE | www.plosone.org 3 September 2008 | Volume 3 | Issue 9 | e3266