Regional Distribution of the NMDA Receptor NR1, NR2A and NR2B Subunits During Moderate Hypoxia in the Newborn Piglet Brain † 1042

Abstract

Characteristics of the NMDA receptor ion-channel are determined by its subunit composition of the NRI and the NR2 subunits. Previous studies have shown that severe brain tissue hypoxia modifies the NMDA receptor ion-channel and its modulatory sites in the cerebral cortex of guinea pigs and newborn piglets. The present study tests the hypothesis that there is a differential regional distribution and response of the NR1, NR2A and NR2B subunits of the NMDA receptor in the newborn piglet brain during moderate hypoxia. Ten anesthetized and ventilated 1-3 day old newborn piglets were divided into normoxic and hypoxic groups. Moderate hypoxia was induced by lowering the FiO2 to achieve PaO2 of 20-25 mmHg. Brain tissue was removed and hippocampus, frontal cortex, temporal cortex, parietal cortex, basal ganglia, hypothalamus, white matter and cerebellum were dissected. P2 membrane fractions were prepared from each of the brain regions. Using specific antibodies to the NR1, NR2A and NR2B subunits the protein was immunoprecipitated, separated on a 7.5% SDS-PAGE and blotted on nitrocellulose paper. The protein bands were developed using a horseradish peroxidase conjugate and visualized with 4 chloro-1-naphthol. The protein bands were analyzed by a imaging densitometer. The results show that the NR1 subunit was distributed differently in the nine specific regions of the newborn piglet brain; hippocampus, frontal cortex > temporal cortex, parietal cortex, thalamus, hypothalamus, basal ganglia > cerebellum, white matter. The NR2A subunit was also distributed differently in the regions; hippocampus, temporal cortex, frontal cortex, parietal cortex, basal ganglia, thalamus > white matter > cerebellum, hypothalamus. Similarly, the NR2B subunit was distributed differently in the nine different sites of the newborn piglet brain; hippocampus, temporal cortex, frontal cortex, parietal cortex, basal ganglia, thalamus > hypothalamus > white matter > cerebellum. In contrast to our previous data where severe hypoxia resulted in decreased expression of NR1, NR2A and NR2B subunits, moderate hypoxia did not alter the expression of these subunits in different brain regions of the newborn piglet. We speculate that the increased distribution of NMDA receptor subunits in the hippocampus and cerebral cortex will contribute to increased vulnerability of these structures to excitotoxicity in the newborn brain.