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

摘要

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-kappaB 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-alpha and IL-1beta both led to pronounced nuclear activation of NF-kappaB in the dentate granule cells as demonstrated by immunostaining and EMSA, respectively. Electrophysiological measurements taken from slices treated with TNF-alpha showed that it inhibited LTP (field excitatory post-synaptic potentials (fEPSP) 116+/-10%, n=9, 60 min post-tetanus compared to control fEPSP 185+/-9%, n=6; P<0.001). The neurotransmitter L-glutamate also led to activation of NF-kappaB and electrophysiology recordings showed a small but sustained increase in synaptic transmission (fEPSP 106+/-12%, 30 min post-drug). These methods provide valuable tools to forward our understanding of the role of NF-kappaB in plasticity as well as in many neurological disorders being mimicked by in vitro studies.