Both neurosteroids and nitric oxide (NO) are neuroactive modulators. The effect of pregnenolone sulfate (PREGS), a representative neurosteroid, on the production of NO in hippocampal slices was investigated with digital fluorescence microscopy using a newly synthesized NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM). Upon stimulation with N-methyl-D-aspartate (NMDA), DAF-FM fluorescence increased to 126%. Preincubation of slices with 100μM PREGS for 20 min increased NMDA-stimulated DAF-FM fluorescence to 148%. Such fluorescence increase was suppressed by both L-NMMA (NO synthase (NOS) inhibitor) and MK-801 (NMDA receptor antagonist). Preincubation with PREGS also enhanced NMDA-induced Ca2+ influx in hippocampal slices, as measured with fura-2. The localization of neuronal NOS protein in pyramidal neurons of the CA1 region was demonstrated using immunohistochemistry combined with in situ blotting of unfixed slices. Taken together, these results imply that PREGS acutely enhances NO production in the hippocampal CA1 neurons, due to an increase in Ca2+ influx through NMDA receptors. Because NMDA-induced acute synthesis of PREGS in hippocampal pyramidal neurons has been demonstrated, a postsynaptic signal amplification circuit including PREGS and NO may function in pyramidal neurons.
Using nanofabrication techniques, we formed artificial nanopore structures on a quartz plate in order to mimic biophysical and biochemical processes. We investigated the dynamics of a long DNA chain (T4 DNA: 166 kbp) passing through the nanofabricated pore (ca.100 nm width) under electric fields (0.5 V-3 V) by means of fluorescence microscopy. The relationship between the applied voltage and the translocation time of DNA is discussed in terms of single chain dynamics. The T4 DNA greatly changed conformation during the translocation process because the pore size was much smaller than the Flory radius of T4 DNA. A characteristic dumbbell structure was observed during the middle stages of the translocation. We point out the importance of this conformational change, since the entropic elasticity affects the translocation dynamics of DNA.