Development of a novel confocal microscopy and analysis of glia-neuron communication

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We constructed a real time scanning confocal microscopy system. This system enables gaining real-time serial confocal images by scanning the objective lens vertically. We carried out following experiments to evaluate its function. Upon mechanical stimulation using glass micropipette, Ca^<2+> signal was generated in a single astroglial cell. Radial propagation of Ca^<2+> wave across cultured astrocytes was observed. The Ca^<2+> wave reached a neuron in the fringe of astrocyte layer. Also Ca^<2+> signal from a mechanically stimulated neuron could elicit Ca^<2+> wave on the astrocyte layers. Thus, we indicated bi-derectional propagation of Ca^<2+> signals between cultured neurons and astrocytes with improved spatial resolution compared to conventional microscopy system. Next, we investigated a modulatory effect of a neurosteroid, pregnenolone sulfate (PREGS), on Ca^<2+> signal mediated by NMDA receptor in CA1 neurons of acute hippocampal slices after 20 min incubation with PREGS. Ca^<2+>-sensitive fluorescent dye Fura-2 was introduced in hippocampal neurons. Intracellular Ca^<2+> rise through NMDA receptor was doubled with 100 M PREGS, leading significant up-regulation of synaptic transmittion efficiency.Also we are studying synthesis of neuroteroids and found that activity-dependent Ca^<2+> signals in hippocampal neurons could facilitate the synthesis of PREGS. Our results revealed a positive feedback system of neural transmittion; Ca^<2+> signal by neural activity induces the synthesis of PREGS from cholesterol→secreted PREGS enhances Ca^<2+> signal by NMDA receptors→facilitation of synaptic transmittion. we are currently proposing a novel concept of local synthesis and acute action of neurosteroids.