Distinct subcellular localization and mRNA expression of neuronal nitric oxide synthase in the nucleus dorsalis and red nucleus and their correlation with inducible transcription factors after spinal cord hemisection

We previously reported on the differential expression of neuronal nitric oxide synthase (nNOS) in neurons of the nucleus dorsalis (ND) and red nucleus (RN), as well as differential roles of nitric oxide (NO) in these two distinct groups' neurons characterized with different nNOS phenotypes after lower thoracic spinal cord hemisection. To further understand the enzyme, nNOS expression was studied at the subcellular and mRNA levels by using electron microscopic immunohistochemistry (EM-IHC) and in situ hybridization respectively. Possible transcriptional regulation by c-Jun or CREB in the differential nNOS expression in both ND and RN neurons was also studied. nNOS mRNA was not found in the normal ND neurons, but was shown in the normal RN neurons. After spinal cord hemisection, nNOS mRNA was induced in the ipsilateral ND, while up-regulated on both sides of the RN, which preceded protein induction or upregulation. By EM-IHC, nNOS immunoreaction products were predominantly bound to the membrane of the mitochondria, rough endoplasmic reticulum (rER), Golgi apparatus, and nuclear envelope in the RN neurons of normal rats as well as rats subjected to spinal cord hemisection. In contrast, nNOS-immunoreactive deposits in the experimental ND neurons were found to be mainly granular, being dispersed throughout the cytoplasmic matrix. It is speculated that the differential subcellular localization of nNOS indicates that axotomy may trigger different nNOS transcripts and lead to different nNOS isoform expression in the normally non-nNOS- and normally nNOS-containing neurons. c-Jun was induced in the ipsilateral ND neurons and upregulated only in the contralateral RN neurons. Activation of CREB by phosphorylation was occasionally detectable in the ND neurons, but not in the RN neurons. Doublelabeling data showed a large proportion of c-Jun and nNOS colocalization in neurons of the ipsilateral ND and contralateral RN after spinal cord hemisection. However, dissociation of nNOS expression kinetics with c-Jun was observed in the ipsilateral RN. The results implied that nNOS expression might not be under the direct transcriptional regulation by c-Jun, although it seemed to be closely related to the c-Jun expression.