Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization

Abstract

The hippocampus consists of a series of cytoarchitecturally discrete subregions that can be distinguished from one another on the basis of morphology, connectivity, and electrophysiological properties. To understand the molecular underpinnings for these differences, DNA microarrays were used to find genes predicted to be enriched in subregions CA1, CA3, and the dentate gyrus, and >100 of these genes were subsequently analyzed using in situ hybridization to obtain cellular-level localization of their transcripts. The most striking commonality among the resulting patterns of gene expression is the extent to which cytoarchitectural boundaries within the hippocampus are respected, although the complexity of these patterns could not have been predicted on the basis of the microarray data alone. Among those genes with expression that can be characterized as "restricted" to neurons in one or more subregions of the hippocampus are a number of signal transduction molecules, transcription factors, calcium-binding proteins, and carbohydrate-modifying enzymes. These results suggest that important determinants of the unique identities of adult hippocampal neurons are differential signal transduction, regulation of gene expression, calcium homeostasis, and the maintenance of a unique extracellular milieu. Furthermore, the extremely high correlation between microarray data and in situ expression demonstrates the great utility of using DNA microarrays to genetically profile discrete brain regions.

Figure 1.

Schematic of microarray data analysis. Data from regions CA1, CA3, and the dentate gyrus of each individual set of animals were grouped together to assess relative differences in gene expression across these regions within a single experiment (A, top panel). Expression levels are plotted as a continuous line for a particular gene, normalized to the median value of all genes on that microarray and to the median value for that gene across all microarrays, and color coded for the expression level of that gene relative to all other genes. Data from all 11 experiments are plotted in the bottom panel in A. B, Data from all 11 independent replicates were probed simultaneously for genes matching a particular pattern of expression (highest in CA1, highest in CA3, or highest in DG). The subset of genes matching this pattern are plotted in C, and one individual gene matching each pattern across all 11 replicates is plotted in D.

Figure 2.

Subregion-specific patterns of gene expression within the hippocampus I. A–E, U–Y, Genes enriched in the dentate gyrus (A–C), CA1–CA3 (D, E), CA3 or CA2 plus CA3 (CA2/3; U–W), and the dentate gyrus plus CA1 (X, Y). A–J, U–DD, In situ hybridization for Dsp (A, F), Mcmd6 (B, G), calretinin (C,H), Lpl (D, I), Mrg1b (E, J), Bok (U,Z) Socs2 (V,AA), Cadps (W,BB), calbindin (X, CC), and Ngef (Y,DD) on coronal sections through the hippocampus at low magnification (A–E, U–Y) and high magnification (F–J, Z–DD). Bisbenzimide staining of the same sections is shown in K–O and EE–II, and gene chip data for each gene are shown in P–T and JJ–NN. Arrowheads in F–O and Z–II delimit the boundaries of CA1 and also the boundary between CA2 and CA3 in Z and EE. The arrow in H denotes the hilus of the dentate gyrus. Scale bars, 1 mm.

Figure 3.

Subregion-specific patterns of gene expression within the hippocampus II. A–D, Q–T, Genes enriched in CA1 (A, B), the dentate gyrus plus CA2/3 (C,D), the dentate gyrus plus CA2 (Q,R), and CA1 plus CA3 (S,T). A–H, Q–X, In situ hybridization for Nov (A, E), Etv1 (B, F), Tgfb2 (C,G), Frzb (D, H), Pcp4 (Q,U), Tiam1 (R, V), Tyro3 (S,W), and Prss19 (T,X) on coronal sections through the hippocampus at low magnification (A–D, Q–T) and high magnification (E–H, U–X). Bisbenzimide staining of the same sections is shown in I–L and Y–BB, and gene chip data for each gene are shown in M–P and CC–FF. Arrowheads in E–L and U–BB delimit the boundaries of CA1 and also the boundary between CA2 and CA3 in U–BB. Scale bars, 1 mm.

Figure 4.

Genes expressed in fimbrial oligodendrocytes, the choroid plexus, and the subiculum. A–G, In situ hybridization for Mobp (A, E), Cldn11 (B, F), Msx1 (C,G), and Fn1 (D) on coronal sections through the hippocampus at low magnification (A–D) and high magnification (E–G). Bisbenzimide staining of the same sections is shown in H–K, and gene chip data for each gene are shown in L–O. Large arrowheads mark the fimbria in E, F, H, and I, arrows in G and J mark the choroid plexus, and arrowheads delimit the subiculum in D and K. Scale bars, 1 mm.

Figure 5.

Summary of expression patterns for genes enriched in hippocampal subregions. Genes are clustered into categories on the basis of their expression in primary excitatory neurons in the dentate gyrus, CA1, CA2, CA3, and, in one case, the dentate hilus. A distinction is made between genes with restricted patterns of expression versus those with enriched patterns. Arrowheads in all schematics denote area CA2.