The long-term objective of this research proposal is to augment our understanding of Pb2+ neurotoxicity and cognitive impairment in children. The hypothesis to be tested is that Pb2+ exposure during development results in a change in the state of phosphorylation of N-methyl-D-aspartate receptor (NMDAR) subunits and this change is related to the impairment of learning and memory. The hippocampus will be removed from rats exposed to 0,750 or 1500 ppm Pb2+-acetate, at post-natal days 14, 21, 28 and 50. Using two-dimensional gel electrophoresis we will separate homogenates of brain tissue according to their isoelectric point and molecular weight. Specific antibodies against the NMDAR -1, 2A and 2B subunits as well as antibodies against phospho-specific residues (tyrosine, serine and threonine) will be used for immunoblot analysis. In vivo long-term potential (LTP) will be induced in the perforant path-dentate gyrus excitatory synapses in 50- day-old control and Pb2+-exposed rats. The dentate gyrus will be subjected to two-dimensional gel electrophoresis to determine the state of phosphorylation of the NMDAR subunits. The contralateral dentate gyrus will be studied as an internal control. To examine whether protein kinase C (PKC) is responsible for changes in phosphorylation we will immunoblot with PKC-phosphospecific NR1 antibodies. PKC-specific phospho-specific phospho-antibodies are not yet available for the NR2A and 2B subunits. Therefore, the subunits will be studied by in vitro back-phosphorylation of hippocampal homogenates using exogenous PKC and [32P]-ATP.
| Nihei, M K; McGlothan, J L; Toscano, C D et al. (2001) Low level Pb(2+) exposure affects hippocampal protein kinase C gamma gene and protein expression in rats. Neurosci Lett 298:212-6 |
