Ruan Hang-Ze, Wang Li-Qing, Yuan Fei, Weng Shi-Jun, Zhong Yong-Mei
Department of Ophthalmology, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science and Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
Department of Ophthalmology, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science and Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
Neuropharmacology. 2021 Apr 1;187:108492. doi: 10.1016/j.neuropharm.2021.108492. Epub 2021 Feb 12.
In this work, modulation by orexin-A of the release of glutamate and GABA from bipolar and amacrine cells respectively was studied by examining the effects of the neuropeptide on miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of rat retinal ganglion cells (GCs). Using RNAscope in situ hybridization in combination with immunohistochemistry, we showed positive signals for orexin receptor-1 (OXR) mRNA in the bipolar cell terminals and those for orexin receptor-2 (OXR) mRNA in the amacrine cell terminals. With whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that application of orexin-A reduced the interevent interval of mEPSCs of GCs through OXR. However, it increased the interevent interval of mIPSCs, mediated by GABA receptors, through OXR. Furthermore, orexin-A-induced reduction of mEPSC interevent interval was abolished by the application of PI-PLC inhibitors or PKC inhibitors. In contrast, orexin-A-induced increase of GABAergic mIPSC interevent interval was mimicked by 8-Br-cAMP or an adenylyl cyclase activator, but was eliminated by PKA antagonists. Finally, application of nimodipine, an L-type Ca channel blocker, increased both mEPSC and mIPSC interevent interval, and co-application of orexin-A no longer changed the mEPSCs and mIPSCs. We conclude that orexin-A increases presynaptic glutamate release onto GCs by activating L-type Ca channels in bipolar cells, a process that is mediated by an OXR/PI-PLC/PKC signaling pathway. However, orexin-A decreases presynaptic GABA release onto GCs by inhibiting L-type Ca channels in amacrine cells, a process that is mediated by an OXR/cAMP-PKA signaling pathway.
在本研究中，通过检测神经肽对大鼠视网膜神经节细胞（GCs）微小兴奋性突触后电流（mEPSCs）和微小抑制性突触后电流（mIPSCs）的影响，分别研究了食欲素-A对双极细胞和无长突细胞释放谷氨酸和γ-氨基丁酸（GABA）的调节作用。利用RNAscope原位杂交结合免疫组化技术，我们在双极细胞终末检测到食欲素受体-1（OXR）mRNA的阳性信号，在无长突细胞终末检测到食欲素受体-2（OXR）mRNA的阳性信号。通过对大鼠视网膜切片进行全细胞膜片钳记录，我们证明应用食欲素-A可通过OXR降低GCs的mEPSCs的事件间期。然而，它通过OXR增加了由GABA受体介导的mIPSCs的事件间期。此外，应用磷脂酰肌醇特异性磷脂酶C（PI-PLC）抑制剂或蛋白激酶C（PKC）抑制剂可消除食欲素-A诱导的mEPSCs事件间期缩短。相反，8-溴环磷酸腺苷（8-Br-cAMP）或腺苷酸环化酶激活剂可模拟食欲素-A诱导的GABA能mIPSCs事件间期延长，但蛋白激酶A（PKA）拮抗剂可消除这种延长。最后，应用L型钙通道阻滞剂尼莫地平可增加mEPSCs和mIPSCs的事件间期，同时应用食欲素-A不再改变mEPSCs和mIPSCs。我们得出结论，食欲素-A通过激活双极细胞中的L型钙通道增加突触前谷氨酸释放到GCs上，这一过程由OXR/PI-PLC/PKC信号通路介导。然而，食欲素-A通过抑制无长突细胞中的L型钙通道减少突触前GABA释放到GCs上，这一过程由OXR/cAMP-PKA信号通路介导。
