The pro-inflammatory cytokine tumour necrosis factor-a upregulates nuclear factor-κb in selective neuronal subpopulations of the rat hippocampus

Graham K. Sheridan, Mark Pickering, Paul N. Moynagh, John J. O’Connor & Keith J. Murphy, UCD School of Biomolecular and Biomedical Science, College of Life Sciences, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

There is evidence to suggest that dysregulations of the immune system may play a major role in modulating certain functions of the central nervous system (CNS) such as learning and memory. Studies of patients with cognitive impairments characteristic of Alzheimer’s disease (AD) have demonstrated elevated levels of pro-inflammatory cytokines in their cerebro-spinal fluid1. Epidemiological studies have also shown a decreased incidence of AD in patients who were being treated with certain non-steroidal anti-inflammatory drugs (NSAIDs) for inflammatory diseases such as rheumatoid arthritis2. Pro-inflammatory cytokines such as TNF-α and IL-1β have been shown to exert an inhibitory effect on long term potentiation (LTP) 3, a form of synaptic plasticity thought to underlie memory formation. One of the main mechanisms by which TNF-α exerts its effects is through the activation of the transcription factor nuclear factor- κB (NF κB). In this study we have investigated the effect of TNF-α on the expression of active NF κB in 5 distinct neuronal subpopulations of the rat hippocampus. P30 (~ 50 grams) male Wistar rats were used in this study. The brain was dissected out and transverse hippocampal slices (400μm) were cut from the dorsal hippocampus. Control slices were incubated in artificial cerebro-spinal fluid (aCSF) at 32 oC for between 0 and 2hrs. Treated hippocampal slices were incubated in aCSF for 90mins, after which 5ng/ml TNF-α was added to the wells and the slices were left to incubate for a further 30mins. The slices were fresh frozen and then cryosectioned at 12μm. Sections were stained with a monoclonal antibody (Mab3026, Chemicon) that binds to the p65 subunit of active NF κB, which in turn was labelled with a FITC-conjugated secondary antibody. Nuclei were stained with propidium iodide. Sections were visualised using a confocal microscope (Zeiss, LSM Pascal) and relative fluorescence intensities for active cytosolic and nuclear NF κB were measured in randomly sampled cells of the CA1, CA2, CA3 and the inner and outer dentate regions of the hippocampus. The ratio of nuclear to cytosolic NF κB was taken as an indicator of nuclear NF κB expression. Data are represented as mean ± s.e.m and analysed using one-way ANOVA. In control slices there was a general trend toward increased levels of active NF κB expression in all examined regions, with relative fluorescence intensities after 2hrs greater than pre-incubation levels (CA1, from 2.05 ± 0.05, n=220 to 2.86 ± 0.11, n=202, p<0.001; CA2, from 2.00 ± 0.11, n=176 to 3.09 ± 0.18, n=164, p<0.001; CA3, from 2.03 ± 0.07, n=196 to 2.93 ± 0.17, n=182, p<0.001; inner dentate, from 1.75 ± 0.07, n=176 to 2.60 ± 0.10, n=184, p<0.001; and outer dentate, from 2.35 ± 0.08, n=192 to 3.20 ± 0.11, n=222, p<0.001). Those slices treated with TNF-α showed increased nuclear NF k B expression compared to their time-matched controls in neurons of the CA1 (3.43 ± 0.13, n=194, p<0.001), inner dentate (3.15 ± 0.16, n=182, p<0.001) and outer dentate regions (3.89 ± 0.15, n=222, p<0.001), but not in the CA2 (3.38 ± 0.15, n=198, p>0.05) or CA3 (3.34 ± 0.16, n=184, p>0.05) neuronal cells.

In summary, we have shown that levels of active nuclear NF κB rise over time in hippocampal slice preparations. We have also demonstrated that the pro-inflammatory cytokine, TNF-α , selectively upregulates NF κB expression in the CA1 and dentate gyrus regions of the hippocampus. This regional specificity must be considered when investigating the role of TNF-α , and possibly other cytokines, in regulating synaptic plasticity.

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This work was supported by Science Foundation Ireland (SFI).