Orexin对视网膜双极细胞功能的调制及其机制

The neuropeptides orexin A/ B are involved in a variety of physiological functions in the central nervous system. Our recent work shows, for the first time, that in the inner retina orexin-A differentially modulates AMPA receptor-mediated current responses of ganglion cells and amacrine cells respectively in rats. In this project we focus on modulation by orexins of activities of bipolar cells, which relay signals from photoreceptors to ganglion cells. Whereas orexins are implicated in regulating circadian rhythms and wake/sleep states, it is reasonable to speculate that orexins may differentially modulate retinal scotopic and photopic functions, which are mediated by rod and cone photoreceptors respectively. We will first investigate how the synthesis and release of orexins in the retina may be dependent on ambient illumination and day/night cycle, and how orexins modulate different components (a, b-waves etc.) of the eletroretinogram (ERG), which reflects neuronal activities in the outer retina, under dark- and light-adapted conditions. Using patch-clamp recording techniques we will further explore whether and how the activity of rod- and cone-driven bipolar cells could be differentially modulated by orexins, based on a detail analysis of orexin-induced changes in transmitter release presynaptic to these cells and current responses of these cells mediated by excitatory and inhibitory receptors, as well as the underlying intracellular mechanisms. This study will therefore provide insight into neuromodulatory role of orexins in the outer retina.

神经活性肽orexin在中枢参与调节多种生理功能。我们最近的研究率先显示在内层视网膜，orexin以不同方式调制无长突细胞和神经节细胞的谷氨酸电流。在本申请中，我们将专注于orexin对外层视网膜中主要的中继神经元——双极细胞（BC）活动的调制。鉴于orexin参与昼夜节律、睡眠/觉醒的调节，我们假设：orexin可能以不同方式调制视网膜分别由视杆和视锥系统介导的暗视（scotopic）和明视（photopic）功能。我们将首先研究orexin在视网膜中的生成和释放的规律，分析orexin对暗视和明视视网膜电图（ERG）（反映外层视网膜总体活动）不同组分(a波和b波等)的调制。在此基础上，运用膜片钳技术研究orexin如何影响分别接受视杆和视锥信号的BC特性，包括BC突触前的递质释放，及所表达的突触后受体介导的电流及其机制。本研究将是首次对orexin在外层视网膜的神经调制作用的研究。

在脑中，食欲肽（orexin）A和B通过激活两个G蛋白偶联受体：OX1R和OX2R在觉醒和摄食过程中起到重要作用。我们先前的研究表明，orexin及其受体在大鼠视网膜神经元中有表达，并且这些肽调制内层视网膜神经元的活动，但其对外层视网膜神经元活动的影响仍不知晓。在本研究中，我们报道：..(1)Orexin B在大鼠外层视网膜中调制视杆双极细胞（RBC）的突触传递.玻璃体腔内注射orexin B使暗视视网膜视图（ERG）b波的幅度增加，暗视ERG b波反应RBC的活动。在视网膜切片上的膜片钳记录显示，orexin B不改变由光感受器驱动的至RBC的谷氨酸兴奋性输入。在视网膜切片标本中，orexin B压抑了内网层中RBC的GABA受体介导的抑制性突触后电流。此外，在分离细胞上的全细胞膜片钳记录显示，orexin B压抑RBC的GABAc受体介导的电流，并且这种抑制作用能被OX1R和OX2R的拮抗剂阻断。orexin B诱导的对GABAc电流的压抑作用经由Gi/o/PC-PLC/Ca2+-independent PKC信号通路介导。这些结果提示，orexin B引起的大鼠视网膜RBC活动的增强或许能改善黑暗期间（觉醒期间）动物的视敏度和对比敏感度。..(2)Orexin-A压抑外层和内层光感受器驱动的至多巴胺无长突细胞（DAC）的信号传递 . Orexin-A减小大多数DAC上视锥/视杆介导的光反应，并抑制所有显示视黑质依赖光反应的DAC，提示orexin能压抑视锥、视杆以及视网膜自感光神经节细胞（ipRGC）向DAC的信号传递。Orexin-A对视黑质介导的光反应的抑制作用是通过影响DAC上的orexin受体实现的，而orexin-A对视锥和视杆至DAC的信号调控则是通过激活DAC及其上游神经元上的orexin受体来实现。结果提示，在哺乳动物视网膜，orexin可通过调节多巴胺系统影响视觉功能。