ABSTRACT: The hippocampus is part of a brain network essential for memory function. Paradoxically, the hippocampus is also the brain structure that is most sensitive to hypoxic-ischemic episodes. Here we show that the expression of genes associated with glycolysis and glutamate metabolism in astrocytes and the coverage of excitatory synapses by astrocytic processes undergo significant decreases in the CA1 field of the monkey hippocampus during postnatal development. Given the established role of astrocytes in the regulation of glutamate concentration in the synaptic cleft, our findings indicate that a developmental decrease in astrocytic processes underlies the selective vulnerability of CA1 during hypoxic-ischemic episodes in adulthood, its decreased susceptibility to febrile seizures with age, as well as contribute to the emergence of selective, adult-like memory function. Regulation of gene expression in the postnatally developing hippocampus might contribute to the emergence of selective memory function. However, the mechanisms that underlie the co-regulation of expression of hundreds of genes in different cell types at specific ages in distinct hippocampal regions have yet to be elucidated. By performing genome-wide microarray analyses of gene expression in distinct regions of the monkey hippocampal formation during early postnatal development, we identified one particular group of genes exhibiting a down-regulation of expression, between birth and six months of age in CA1 and after one year of age in CA3, to reach expression levels observed at 6-12 years of age. Bioinformatics analyses using NCBI, miRBase, TargetScan, microRNA.org and Affymetrix tools identified a number of miRNAs capable of regulating the expression of these genes simultaneously in different cell types, i.e., in neurons, astrocytes and oligodendrocytes. Interestingly, sixty-five percent of these miRNAs are conserved across species, from rodents to humans; whereas thirty-five percent are specific to primates, including humans. In addition, we found that some genes exhibiting greater down-regulation of their expression were the predicted targets of a greater number of these miRNAs. In sum, miRNAs may play a fundamental role in the co-regulation of gene expression in different cell types. This mechanism is partially conserved across species, and may thus contribute to the similarity of basic hippocampal characteristics across mammals. This mechanism also exhibits a phylogenetic diversity that may contribute to more subtle species differences in hippocampal structure and function observed at the cellular level. Sixteen male rhesus monkeys (Macaca mulatta; four 1-day-olds, four 6-month-olds, four 1-year-olds and four 6-12-year-olds) were used for this study. Monkeys were injected with an overdose of sodium pentobarbital (50 mg/kg i.v., Fatal-Plus, Vortech Pharmaceuticals, Dearborn, MI) and the brain rapidly extracted. Five-millimeter thick slices of the brain were cut and stored overnight in RNAlaterä (Ambion, Austin, TX) at 4oC. Brain slices were then frozen in liquid nitrogen and re-sectioned at 100 µm for microdissection. Five hippocampal regions were microdissected, including the entorhinal cortex (all layers at the mid-rostrocaudal level; intermediate division, Ei), and all layers of the dentate gyrus, CA3, CA1 and subiculum at mid-rostrocaudal level of the body of the hippocampus (at the level of the lateral geniculate nucleus). Only the mid-transverse portion of each region was dissected to ensure the specificity of the sample. RNA was isolated with Trizol® and single stranded cDNA synthesized starting with 10 µg of total RNA. The sample from each region from each monkey was run on a separate chip, thus totaling 4 (animals per age) X 4 (ages) X 5 (regions) = 80 independent chips.
