Distribution and quantification of NMDA R1 mRNA and protein in the piglet brainstem and effects of intermittent hypercapnic hypoxia (IHH).

The first aim of this study was to determine the cellular distribution of NR1 in the piglet brainstem. Documentation of NR1 mRNA was by non-radioactive in situ hybridisation (non-RISH) and of NR1 protein by immunohistochemistry. We found that all neurons expressed mRNA but not all had NR1 protein. Application of non-RISH has permitted us, for the first time, to document the cellular localization of NR1 mRNA showing that it was present in the cytoplasm and nucleolus of motor neurons but dispersed throughout the cellular compartments of sensory neurons. NR1 protein on the other hand, was localized to the cytoplasm only. The second aim of this study was to quantify the distribution of NR1 mRNA and protein. Using image analysis software, we used optical density (OD) to quantify the non-RISH signal for mRNA and cellular counting for protein (expressed as % positive). Results show that NR1 expression is greater in motor than sensory nuclei; for mRNA: 0.46+/-0.04 vs. 0.31+/-0.02 OD units (P<0.001), for protein: 75.9+/-3.1 vs. 58.4+/-2.5% positive (P<0.001). The third aim of this study was to determine the effects of intermittent hypercapnic hypoxia (IHH) on NR1 expression. Chronic IHH exposure caused differential changes in mRNA and protein expression. NR1 mRNA expression increased while the number of neurons expressing NR1 protein showed no change. This finding suggests that NMDA pathways are activated by exposure to IHH.