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

We previously reported on the differential expression of neuronal nitric ox ide 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 dist inct groups' neurons characterized with different nNOS phenotypes after low er thoracic spinal cord hemisection. To further understand the enzyme, nNOS expression was studied at the subcellular and mRNA levels by using electro n microscopic immunohistochemistry (EM-IHC) and in situ hybridization respe ctively. Possible transcriptional regulation by c-Jun or CREB in the differ ential nNOS expression in both ND and RN neurons was also studied. nNOS mRN A was not found in the normal ND neurons, but was shown in the normal RN ne urons. After spinal cord hemisection, nNOS mRNA was induced in the ipsilate ral ND, while upregulated on both sides of the RN, which preceded protein i nduction or upregulation. By EM-IHC, nNOS immunoreaction products were pred ominantly bound to the membrane of the mitochondria, rough endoplasmic reti culum (rER), Golgi apparatus, and nuclear envelope in the RN neurons of nor mal 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 i s speculated that the differential subcellular localization of nNOS indicat es that axotomy may trigger different nNOS transcripts and lead to differen t nNOS isoform expression in the normally non-nNOS- and normally nNOS-conta ining neurons. c-Jun was induced in the ipsilateral ND neurons and upregula ted only in the contralateral RN neurons. Activation of CREB by phosphoryla tion was occasionally detectable in the ND neurons, but not in the RN neuro ns. Double-labeling data showed a large proportion of c-Jun and nNOS coloca lization 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 expr ession might not be under the direct transcriptional regulation by c-Jun, a lthough it seemed to be closely related to the c-Jun expression. (C) 2000 A cademic Press.