Data_Sheet_3_High-Throughput Functional Characterization of Visceral Afferents by Optical Recordings From Thoracolumbar and Lumbosacral Dorsal Root Ganglia.docx

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, Ca2+ indicators like GCaMP6f allow functional characterization by optical recordings. Here we report a turnkey microscopy system that enables simultaneous Ca2+ 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.

内脏传入神经的功能解析，对于开发针对内脏高敏感性与疼痛的新型治疗方案具有重要意义。背根神经节（dorsal root ganglia, DRGs）内内脏传入神经的稀疏分布，给传统电生理记录技术带来了显著挑战。作为替代方案，钙指示剂（Ca²⁺ indicators）如GCaMP6f可通过光学记录实现神经组织的功能表征。本文报道了一款开箱即用型显微镜系统，可对完整背根神经节实现双平行焦平面同步钙成像。该系统采用消费级光学元件搭建，兼具成本效益与普适性，且不会牺牲成像性能。其可在1.9 × 1.3 mm的宽视场（field of view）下采集低强度荧光信号，以覆盖整只小鼠的背根神经节，同时具备0.7 微米/像素的高像素分辨率、50 帧/秒的快速帧率，以及无需扰动样本即可实现远程聚焦（remote focusing）的能力。其电动载物台（motorized sample stage）拥有100 mm的宽扫描范围，可便捷记录胸椎、腰椎与骶椎区域的多个背根神经节。作为应用演示，我们在由VGLUT2启动子驱动表达GCaMP6f的小鼠体内，对支配远端结肠与直肠（colorectum）的内脏传入神经的机械神经编码特征进行了表征。此外，我们开发了一套后处理流程，可从GCaMP6f的成像记录中无监督检测内脏传入神经的响应，并补偿由结肠直肠机械刺激引发的运动伪影（motion artifacts）。本系统为从大量背根神经节组织中高通量记录内脏传入神经活动提供了一种高性价比的解决方案。我们预期该显微镜系统将得到广泛应用，以加速我们对内脏神经支配的功能解析。