Analysis of Distributions of Nitric Oxide Synthase mRNAs in the Normal Rat Brain by In Situ Hybridization Histochemistry

Summary

Distributions of neuronal nitric oxide synthase (n-NOS) and inducible nitric oxide synthase (i-NOS) mRNAs in the normal adult rat brain were investigated by in situ hybridization histochemistry. On examination of n-NOS mRNA, signals were detected in various regions of the cerebrum, the brainstem, and the cerebellum in their constituent neurons. The quantitative analysis revealed that the numbers of signals per cell were variable, ranging from 5–10 to more than 200, depending on the region. Brain regions without distinct n-NOS signals were also seen. This marked difference in the amount of n-NOS transcripts seemed to indicate differences of contribution of NO in physiological function among neurons of different regions. However, as seen in the cerebral cortex, the caudate/putamen, the hippocampal gyrus, and the dentate gyrus, although most neurons showed no or weak signals, a small number of sporadically scattered neurons showed intense signals, indicating functional heterogeneity of neurons even in the same region. Nonneuronal elements including glial cells and endothelial cells revealed no distinct signals for n-NOS mRNA. On examination of i-NOS mRNA, no distinct signals were detected in the brain parenchyma, the choroid plexus, the meninges, or the blood vessels, suggesting that if i-NOS mRNA was expressed in some cells in the normal rat brain, it might be only in a small amount. These findings might provide a basis for studies on regulation of NOS expression at the transcriptional and posttranscriptional levels in various physiological and pathological states, including brain ischemia. Advantages of the use of pyronin-methylgreen in counterstaining and in determination of the optimal conditions of in situ hybridization procedures are also described.