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

我们构建了一个实时扫描共聚焦显微镜系统。该系统可以通过垂直扫描物镜获得实时串行共聚焦图像。我们进行了以下实验来评估其功能。在玻璃微管机械刺激下，单个星形胶质细胞产生Ca^<2+>信号。观察到Ca^<2+>波在培养的星形胶质细胞间的径向传播。Ca^<2+>波到达星形胶质细胞层边缘的神经元。机械刺激神经元的Ca^<2+>信号也可在星形胶质细胞层上引起Ca^<2+>波。因此，我们发现与传统显微镜系统相比，Ca^<2+>信号在培养的神经元和星形胶质细胞之间的双向传播具有更高的空间分辨率。接下来，我们研究了神经类固醇孕烯醇酮硫酸盐（PREGS）对急性海马片CA1神经元中NMDA受体介导的Ca^<2+>信号在PREGS作用20分钟后的调节作用。海马神经元中引入Ca^<2+>敏感荧光染料Fura-2。100 M PREGS使细胞内Ca^<2+>通过NMDA受体升高两倍，导致突触传递效率显著上调。我们也在研究类神经细胞的合成，发现海马神经元中活动依赖性的Ca^<2+>信号可以促进PREGS的合成。我们的研究结果揭示了一个正反馈的神经传递系统；Ca^<2+>信号通过神经活动诱导胆固醇合成PREGS→分泌的PREGS通过NMDA受体增强Ca^<2+>信号→促进突触传递。我们目前提出了神经类固醇局部合成和急性作用的新概念。

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.