Abstract
Aging is characterized by an increased risk of cognitive deficits. It was shown using rodents that aging is associated with a significant reduction in the function of hippocampus, the brain structure important in forming spatial memories. Long-term potentiation (LTP) is an electrophysiologically induced phenomenon, whereby high frequency currents, artificially passed across specific axonal pathways, elicit a long-lasting change in the activity of the postsynaptic neurone. LTP induced in vivo in the adult rat hippocampus can last for months. Interestingly, LTP and learning activate common molecular mechanisms, which when impeded, can affect LTP persistence and memory. Thus, LTP serves as an excellent experimental model for studying memory-related mechanisms. It was previously established that LTP consists of distinct temporal phases, and that LTP-related gene expression changes in the early phase influence the late phase of LTP. Specifically, the robust expression of important immediate early gene transcription factors such as JunB, Fos and zif/268, have been previously shown to be rapidly increased at 20 min post-LTP in vivo; the expression of these factors correlate with LTP persistence. As LTP persistence was found to be curtailed in aged rats (without affecting early LTP), we hypothesised that an age-related alteration in gene expression profiles at soon after LTP induction must contribute to this.
In order to investigate this, adult (4.8 ± 0.14 months; average ± s.e.m, n = 23), middle-aged (14.7 ± 0.3 months; n = 9) and aged (22 months; n = 12) rats that had been behaviourally characterised using the Morris Water Maze, were stimulated unilaterally to induce LTP at the perforant path synapses. This resulted in robust potentiation of field potentials (fEPSP) at 20 min post-LTP (% change in fEPSP for adult: 25.6 ± 6.9; middle-aged: 19.8 ± 0.8; aged: 31.4 ± 5.2), which, as expected, was not significantly different between age groups (one-way ANOVA: p = 0.73). Rats were decapitated at 20 min post-LTP, dentate gyri dissected and total RNA isolated from control and LTP-stimulated dentate gyri. LTP-related gene expression profiles in adult (n = 5), middle-aged (n = 3) and aged (n = 5) rats were investigated using CombRat 27K oligonucleotide microarrays. Data normalisation and statistical analyses were carried out using the TM4 microarray analysis package.
Statistically significant LTP-related gene expression changes in the adult group were first analysed using the Database for Annotation, Visualisation, and Integrated Discovery (DAVID), which revealed a distinct over-representation of transcription-related genes. When the fld changes of these genes were compared to the middle-aged and aged groups, a significant age-related attenuation of up- (Kruskall-Wallis statistic = 13.02; p = 0.002; n = 19 genes) and down-regulation (Kruskall-Wallis statistic = 6.94; p = 0.03; n = 28 genes) was noted (average fold upregulation for adult: 1.43 ± 0.17; middle-aged: 1.32 ± 0.24; aged: 1.18 ± 0.09 and average fold downregulation for adult: 0.73 ± 0.08; middle-aged: 0.8 ± 0.08 and aged: 0.83 ± 0.12).
To establish LTP-related molecular pathways altered with aging, LTP-related genes from each age group (from t-tests) were combined and analysed using an ANOVA. This identified 346 LTP genes altered with aging, which were analysed for over-represented cellular and molecular pathways using Ingenuity Pathway Analysis (IPA). Canonical pathway analysis in IPA showed that known LTP-related mechanisms such as glucocorticoid receptor signalling, cAMP-mediated signalling, estrogen signalling and G-protein coupled signalling were altered with aging. Additionally, novel pathways such as retinoid receptor signalling, hypoxia-induced factor 1α signalling and folate-mediated one-carbon metabolism (one carbon pool by folate) were associated with LTP, and altered with aging. Functional analysis in IPA revealed that cellular and molecular processes pertaining to neurogenesis were associated with LTP, and altered with aging.
Finally, network analysis in IPA showed that LTP-related genes representing different pathways and functions may influence the expression of each other. Importantly, it revealed that NF-κB, histone h3, platelet-derived growth factor BB (PDGFBB), ERK/MAPK and the microRNA, miR124-mediated signalling were altered with aging, even though these molecules were not identified as regulated. A synthesis of data analysis from DAVID and IPA showed that aging may be associated with alterated LTP-related mechanisms involving transcriptional control, neurogenesis and neuroprotection. Additionally, mechanisms leading to neurodegeneration through impairment of endoplasmic reticulum function also appear to be enhanced in aged rats following LTP. Such alterations may not only underlie curtailed LTP persistence, but may also lead to neurodegenerative disorders such as the Alzheimer’s disease.