Hirai Yasuharu, Nishino Eri, Ohmori Harunori
Department of Neurobiology and Physiology, Faculty of Medicine, Kyoto University, Kyoto, Kyoto, Japan; and Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Kyoto, Kyoto, Japan.
Department of Neurobiology and Physiology, Faculty of Medicine, Kyoto University, Kyoto, Kyoto, Japan; and.
J Neurophysiol. 2015 Jun 1;113(10):3930-42. doi: 10.1152/jn.00005.2015. Epub 2015 Mar 11.
Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices.
尽管多光子显微镜高分辨率成像在体内记录神经元信号方面应用广泛，但由于光穿透有限，其仅限于脑组织表面。此外，大多数成像研究并未同时记录神经电活动，而这对于理解脑功能至关重要。因此，我们开发了一种光度膜片电极（PME），以克服光学测量的深度限制，并能够同时记录深部脑区的神经电反应。PME记录系统使用膜片电极激发荧光染料，并将荧光信号作为光导进行测量，以记录电信号，并向记录的细胞局部施加化学物质。根据反应动力学，光学信号由高光灵敏度光谱仪或光电倍增管进行分析。我们在体内将PME用于加载了 Oregon Green BAPTA-1 AM的鸟类听觉核团，以监测钙信号和电反应。我们在听觉上行通路的三个不同核团中展示了不同的反应模式。在声刺激下，听觉皮层（L区）神经元出现强烈的钙荧光反应，该反应持续时间超过电反应。在听觉中脑（下丘），两种反应均为瞬态。在脑干蜗神经核大细胞部，钙反应似乎被代谢型谷氨酸受体的活性有效抑制。总之，PME为在传统成像设备无法到达的组织深度研究体内脑功能提供了一个强大的工具。
