High-throughput functional characterization of visceral afferents by optical recordings from thoracolumbar and lumbosacral dorsal root ganglions

Functional understanding of visceral afferents is important for developing new treatment to visceral hypersensitivity and pain. The sparse distribution of visceral afferents in dorsal root ganglions (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 x 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 artefacts 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 in both health and diseases.