The functional genome of CA1 and CA3 neurons under native conditions and in response to ischemia

Abstract

Background: The different physiological repertoire of CA3 and CA1 neurons in the hippocampus, as well as their differing behaviour after noxious stimuli are ultimately based upon differences in the expressed genome. We have compared CA3 and CA1 gene expression in the uninjured brain, and after cerebral ischemia using laser microdissection (LMD), RNA amplification, and array hybridization.

Results: Profiling in CA1 vs. CA3 under normoxic conditions detected more than 1000 differentially expressed genes that belong to different, physiologically relevant gene ontology groups in both cell types. The comparison of each region under normoxic and ischemic conditions revealed more than 5000 ischemia-regulated genes for each individual cell type. Surprisingly, there was a high co-regulation in both regions. In the ischemic state, only about 100 genes were found to be differentially expressed in CA3 and CA1. The majority of these genes were also different in the native state. A minority of interesting genes (e.g. inhibinbetaA) displayed divergent expression preference under native and ischemic conditions with partially opposing directions of regulation in both cell types.

Conclusion: The differences found in two morphologically very similar cell types situated next to each other in the CNS are large providing a rational basis for physiological differences. Unexpectedly, the genomic response to ischemia is highly similar in these two neuron types, leading to a substantial attenuation of functional genomic differences in these two cell types. Also, the majority of changes that exist in the ischemic state are not generated de novo by the ischemic stimulus, but are preexistant from the genomic repertoire in the native situation. This unexpected influence of a strong noxious stimulus on cell-specific gene expression differences can be explained by the activation of a cell-type independent conserved gene-expression program. Our data generate both novel insights into the relation of the quiescent and stimulus-induced transcriptome in different cells, and provide a large dataset to the research community, both for mapping purposes, as well as for physiological and pathophysiological research.

Figure 1

Experimental model and strategy. Male C57/bl6 mice were subjected to a combined ischemia/hypoxia model which consistently yields full hemispheric infarcts. A, TTC-stained coronal section 24 h after induction of ischemia/hypoxia demonstrate a full hemispheric infarct which fully covers the hippocampal region. B, The cutting outlines used for Laser microdissection from coronal cryosections are demonstrated on a section stained with an antibody against NeuN and a secondary Cy3-coupled antibody. Actual Laser microdissections in the experiments were performed on Thionin-stained sections. C, Scheme showing the strategy used for detection of differentially regulated genes from amplified RNA. Samples from CA3 and CA1 regions were hybridized on two-color oligonucleotide arrays (Agilent). Direct competitive hybridizations were performed for all combinations: CA3 sham vs, CA1 sham, CA3 ischemia vs CA1 ischemia, CA3 ischemia vs CA3 sham, and CA1 ischemia vs CA1 sham. All experiments were also dye-swapped, and means of the two corresponding values used for further analyses. Arrays were statistically analyzed using linear modelling (limma, R).

Figure 2

CA1 and CA3 neurons display a different gene expression repertoire in the native state. Result of the comparison of CA3 and CA1 regions in the native animal. A, We detect 1035 genes that are differentially expressed in the CA3 and CA1 region in sham-treated animals, 566 are significantly enriched in CA3, 487 in CA1 (p_fdr < 0.05; differentially regulated genes in red). B, Bar graph showing enrichment factors for the 5 most differentially expressed genes in CA3 and CA1. The enrichment factors reflect relative abundance of gene expression in CA3 over CA1 or vice versa, and were calculated by dividing the mean of the gene abundance in CA3 by CA1 or vice versa. Bok, Bcl-2 related ovarian killer; pvrl3, poliovirus receptor-related 3; rerg, RAS-like, estrogen-regulated; dehal1, Iodotyrosine dehalogenase 1; mpped1, metallophosphoesterase domain containing 1; penk1, preproenkephalin 1.

Figure 3

Gene ontology analysis for CA3- and CA1-enriched genes in the native state. Bar graph depicting gene ontology (GO) analysis of the genes significantly overexpressed in CA3 or CA1. The upper part of the graph shows the GO category "Biological process", the lower part "Molecular function". GO analysis were performed using the web-based L2L tool [29]. For "Biological process" there is a strong emphasis on terms related to neuron differentiation, synaptic function, and energy metabolism in CA3, and to GABA-signaling in CA1. For "Molecular function" we find enrichment of groups like ephrin receptors, serin-type protease inhibitors, and carbohydrate moiety transfer activities. In contrast in CA1 we observe an enrichment in deglycosylating activity, ligand-gated ion channels, and sulfotransferase activity. Black, expected matches; red, observed matches.

Figure 4

The Bok-protein is localized to CA3. Micrographs showing immunohistochemical detection of Bok (Bcl-2 related ovarian killer) protein expression in the hippocampus of C57/bl6 mice. A, Overview of the hippocampal formation (original magnification (OM) 10×). Bok preferentially localizes to th CA3 subfield, with some staining in the dentate gyrus. Apart from the hippocampus stining is visible in the cortex. B,C,D,E Staining in the CA3 subfield (B), in the CA3-CA2 transition (C), in the CA1 field (D), and in the CA1-subiculum transition (E) (OM 20×). In CA2 only few single neurons are detectable that stain positive for Bok, in CA1 staining is absent and is evident again in the subiculum. F, Close-up of CA3 neurons (OM 60×). Although quite specifically localized to CA3 Bok displays a patchy staining of neurons within this subfield.

Figure 5

Gene expression in CA3 versus CA1 after ischemia. A,B Scatterplots showing the results of direct competitive hybridizations of both subfields from ischemic animals and the non-ischemic controls (means of n = 4 experiments for each region). In each of the hippocampal subregions more than 5000 genes could be identified exhibiting differential expression upon ischemia. A. 5243 differentially expressed genes were detected in the CA3 region. B. 5511 differentially expressed genes were detected in the CA1 region. Red: significantly regulated genes (p_fdr < 0.05; differentially regulated genes in blue). C, A total of 97 genes is significantly different between ischemic CA3 and ischemic CA1 regions (scatterplot; blue are significantly regulated genes p_fdr < 0.05). D, Bar graph showing the 5 most different genes with preference for CA3 or CA1 with their relative enrichment factors (prdma, pr-domain containing protein 8; Inhba, Inhibin beta A; Bok, Bcl-2 related ovarian killer; Sytl4, synaptotagmin-like 4; rbp 4, retinol binding protein 4; mpped1, metallophosphoesterase domain containing 1; mrg1, myeloid ecotropic viral integration site-related gene 1, alternative names: meis2, stra10).

Figure 6

Ischemia blunts the gene expression differences in CA3 and CA1. A, Box-Whisker plot showing the narrowing of CA3/CA1 ratios in ischemic vs. naive animals. B, Plotting CA3/CA1 ratios in ischemia vs. sham reveals deviation from the diagonal. Difference ratios are attenuated by ischemia regardless of preferential CA3 or CA1 expression (red: p_fdr < 0.05). C, Inhibin beta A displays the highest change in preference between the native and ischemic state. The gene expression preference actually reverses from higher expression in CA1 in the native brain to higher expression in CA3 in the ischemic state. Verification with quantitative PCR shows comparable values to the array-derived ratios.