Bian Zichao, Guo Tiantian, Jiang Shaowei, Chen Longtu, Liu Jia, Zheng Guoan, Feng Bin
Department of Biomedical Engineering, University of Connecticut, Mansfield, CT, United States.
Front Neurosci. 2021 Mar 11;15:657361. doi: 10.3389/fnins.2021.657361. eCollection 2021.
Functional understanding of visceral afferents is important for developing the new treatment to visceral hypersensitivity and pain. The sparse distribution of visceral afferents in dorsal root ganglia (DRGs) has challenged conventional electrophysiological recordings. Alternatively, Ca indicators like GCaMP6f allow functional characterization by optical recordings. Here we report a turnkey microscopy system that enables simultaneous Ca imaging at two parallel focal planes from intact DRG. By using consumer-grade optical components, the microscopy system is cost-effective and can be made broadly available without loss of capacity. It records low-intensity fluorescent signals at a wide field of view (1.9 × 1.3 mm) to cover a whole mouse DRG, with a high pixel resolution of 0.7 micron/pixel, a fast frame rate of 50 frames/sec, and the capability of remote focusing without perturbing the sample. The wide scanning range (100 mm) of the motorized sample stage allows convenient recordings of multiple DRGs in thoracic, lumbar, and sacral vertebrae. As a demonstration, we characterized mechanical neural encoding of visceral afferents innervating distal colon and rectum (colorectum) in GCaMP6f mice driven by VGLUT2 promotor. A post-processing routine is developed for conducting unsupervised detection of visceral afferent responses from GCaMP6f recordings, which also compensates the motion artifacts caused by mechanical stimulation of the colorectum. The reported system offers a cost-effective solution for high-throughput recordings of visceral afferent activities from a large volume of DRG tissues. We anticipate a wide application of this microscopy system to expedite our functional understanding of visceral innervations.
对内脏传入神经的功能理解对于开发治疗内脏超敏反应和疼痛的新方法至关重要。内脏传入神经在背根神经节（DRG）中的稀疏分布对传统的电生理记录提出了挑战。相比之下，像GCaMP6f这样的钙指示剂可以通过光学记录进行功能表征。在这里，我们报告了一种交钥匙显微镜系统，该系统能够从完整的DRG在两个平行焦平面上同时进行钙成像。通过使用消费级光学组件，该显微镜系统具有成本效益，并且可以广泛提供而不损失性能。它在宽视野（1.9×1.3毫米）下记录低强度荧光信号，以覆盖整个小鼠DRG，具有0.7微米/像素的高像素分辨率、50帧/秒的快速帧率以及在不干扰样品的情况下进行远程聚焦的能力。电动样品台的宽扫描范围（100毫米）允许方便地记录胸段、腰段和骶段椎骨中的多个DRG。作为演示，我们在由VGLUT2启动子驱动的GCaMP6f小鼠中表征了支配远端结肠和直肠（结肠直肠）的内脏传入神经的机械神经编码。开发了一种后处理程序，用于对GCaMP6f记录中的内脏传入神经反应进行无监督检测，该程序还补偿了结肠直肠机械刺激引起的运动伪影。所报道的系统为从大量DRG组织中高通量记录内脏传入神经活动提供了一种经济高效的解决方案。我们预计这种显微镜系统将得到广泛应用，以加快我们对内脏神经支配的功能理解。
