Pregnenolone Sulfate Acutely Enhances NO Production in the Rat Hippocampus: Digital Fluorescence Study using NO Reactive Dye

Abstract

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.