Calcium Ions In Nerve Cells And Nitric Oxide Dual-labeling Method And Applications

Ca~2+ and Nitric Oxide(NO) are important messenger molecules in neurons. They play crucial roles in the regulation of numerous physiological and pathological processes in central neuvous system through various signal pathways. There is close inter-regulation between Ca~2+ and NO in neurons. To simultaneously detect the intracellular Ca~2+ and NO in neurons will be helpful to the study of their interrelationship.In this study, choosing 8-10 days cultured hippocampal neurons of SD rat, applying Laser Scanning Confocal Microscope (LSCM), we proposed simultaneous detection of intracellular Ca~2+ and NO by double-label method, based on visible light excitation. This method chooses Calcium Orange AM and DAF-FM diacetate as the fluorescent indicators of intracellular Ca~2+ and NO respectively. 'Two Track' mode of LSCM is applied to realize simultaneous detection of intracellular Ca~2+ and NO through quickly switching excitation wavelengths. The experiment results show that there is no cross talk between the two dyes and the double-label method can reveal the changes of intracellular Ca~2+ and NO concentrations under the stimulation of N-methyl-D-aspartate (NMDA), consistent with the results of respective single-label experiments. So we consider that the double-label method is practicable and credible.Using this double-label method, we did some primary study about the distribution of intracellular Ca~2+ and NO in cultured hippocampal neurons and their changes under NMDA stimulation. The experiment results reveal that the distributions of intracellular Ca~2+ and NO are quite consistent; the concentration of Ca~2+ in nuclear region is obvious higher than that in plasma and so is the concentration of NO; the distribution of Ca~2+ and NO in nuclear and plasma is not uniform and seem to congregate in some netlike structures. After NMDA stimulation, he concentrations of Ca~2+ and NO in both nuclear and plasma rise observably, especially in the netlike structures; the change of Ca~2+ concentration in nuclear is larger than that in plasma,however the changes of NO concentration in nuclear and plasma are similar. We also observed comparative increases of the concentration of Ca2+ and NO in neurodendrite after NMDA stimulation.Our study suggests that the double-label method can simultaneously detect the intracellular Ca2+ and NO in cultured hippocampal neurons and thus provide a helpful approach to investigate the interaction between Ca2+ and NO in neurons.