Methods of detection of the transcription factor NF-κB in rat hippocampal slices

Abstract

The hippocampus is one of the most studied sites for understanding the cellular and molecular mechanisms underlying long-term potentiation (LTP) and long-term depression (LTD), mechanisms believed to underlie the formation and storage of memories. The early-phases of LTP and LTD have been most intensively studied and have been shown to involve the activation of several kinases and phosphatases, respectively. The factors involved in the later stages have largely yet to be elucidated. We have focused our attention on the transcription factor NF-kB as a possible factor involved in such late-phase processes, and have developed both immunocytochemistry and electrophoretic mobility shift assay (EMSA) to measure the activated forms of this factor. This is important as many of the studies in this area are performed in vitro and to our knowledge quantitative assessment has not previously being deemed feasible in slice work. The pro-inflammatory cytokines TNF-a and IL-1b both led to pronounced nuclear activation of NF-kB in the dentate granule cells as demonstrated by immunostaining and EMSA, respectively. Electrophysiological measurements taken from slices treated with TNF-a showed that it inhibited LTP (field excitatory post-synaptic potentials (fEPSP) 1169/10%, n 0/ 9, 60 min post-tetanus compared to control fEPSP 1859/9%, n 0/ 6; P B/0.001). The neurotransmitter L-glutamate also led to activation of NF-kB and electrophysiology recordings showed a small but sustained increase in synaptic transmission (fEPSP 1069/12%, 30 min post-drug). These methods provide valuable tools to forward our understanding of the role of NF-kB in plasticity as well as in many neurological disorders being mimicked by in vitro studies. #

Deletion of Nuclear Factor kappa B p50 Subunit Decreases Inflammatory Response and Mildly Protects Neurons from Transient Forebrain Ischemia-induced Damage

“…Neuronal cell loss and inflammation in the hippocampus have previously been shown to significantly impair synaptic plasticity and spatial learning [28,[63][64][65][66]. In our current study, we did not detect a clear effect of BCCAo on spatial learning and memory most likely due to both small average lesion size and advanced age of the animals (11-month-old on average).…”

Dissection of tumor-necrosis factor-α inhibition of long-term potentiation (LTP) reveals a p38 mitogen-activated protein kinase-dependent mechanism which maps to early—but not late—phase LTP

“…In some slices, this potentiation lasted for up to 6 h (data not shown). When TNF-␣ (4.5 ng/ml) was applied to the hippocampal slice 20 min pre-tetanus early-LTP (TNF-␣/LTP 120Ϯ7%, nϭ6 versus control LTP 175Ϯ6%, nϭ6 1 h post-tetanus, PϽ0.001) was significantly impaired as previously published (Butler et al, 2002). The LTP was followed in these TNF-␣ treated slices for 3 h post-tetanus corresponding to late LTP, and the fEPSP recordings were seen to decrease below baseline fEPSP values and plateaued at approximately 3 h post-tetanus (TNF-␣/LTP fEPSP 68Ϯ5%, nϭ6 versus control LTP 175Ϯ7%, nϭ6, PϽ0.001).…”

Rosiglitazone attenuates the age-related changes in astrocytosis and the deficit in LTP

“…These data suggest that TNF-␣ may play a key role in inducing the observed age-related changes, and because it has been shown to impair LTP in vitro (Butler et al, 2002;Cumiskey et al, 2007;Pickering et al, 2005;Stellwagen and Malenka, 2006;Tancredi et al, 1992) but not, to date in vivo, we assessed its effect on LTP in perforant path-granule cell synapses in urethane-anesthetized rats. The data show that it significantly reduced the ability of rats to sustain LTP (p Ͻ 0.001; Fig.…”