Nitric oxide spatial distribution in single cultured hippocampus neurons: investigation by projection of reconstructed 3-D image and visualization technique.

Recent studies have revealed a non-homogeneous distribution of nitric oxide synthase (NOS) in neurons. However, it is not yet clear whether the intracellular distribution of NOS represents the intracellular nitric oxide (NO) distribution. In the present study, software developed in our laboratory was applied to the reconstructed image obtained from confocal slice images in order to project the 3-D reconstructed images in any direction and to cut the neuron in different sections. This enabled the spatial distribution of NO to be visualized in any direction and section. In single neurons, NO distribution was seen to be heterogeneous. After stimulation with glutamate, the spatial changes in different areas of the neuron were different. These findings are consistent with immunocytochemical data on the intracellular localization of nNOS in hippocampus neurons, and will help to elucidate the specificity of nitric oxide signaling. Finally, the administration of SNAP and L-NAME was used to examine DAF-2 distribution in the neurons. The results showed this distribution to be homogenous; therefore, it did not account for the NO distribution results.

The influence of oxygen-glucose deprivation on nitric oxide and intracellular Ca(2+) in cultured hippocampal neurons.

Nitric oxide (NO) was speculated to play an important role in the pathophysiology of cerebral ischemia. In this study, the effect of oxygen-glucose deprivation (OGD) on the cellular production of NO was investigated in cultured hippocampal neurons. Intracellular Ca(2+) was also detected as its closely relationship with NO. The generation of NO and changes in intracellular Ca(2+) were evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2 DA), an NO probe, and Fluo-3, a Ca(2+) probe respectively. Extracellular glutamate level was also measured by HPLC with fluorescence detection. Results showed that OGD induced an increase in NO production and intracellular Ca(2+) concentration ([Ca(2+)](i)), the rise of DAF-2 and Fluo-3 fluorescence intensity was about 160% and 270% respectively; an increase of about 100% in glutamate level was observed after 20 min of OGD. NMDA inhibitor MK-801 significantly reduced the OGD-induced elevation of [Ca(2+)](i) and NO, DAF-2 and Fluo-3 fluorescence intensity uptake was inhibited by 69% and 74% respectively. The increase in NO production was also attenuated by extracellular Ca(2+) elimination and calmodulin (CaM) antagonist trifluoperazine dose-dependently. These results indicated that NO production increased during oxygen-glucose deprivation, and was greatly modulated by glutamate release, intracellular Ca(2+) change and Ca(2+)-CaM pathway.