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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 2+ indicators like GCaMP6f allow functional characterization by optical recordings. Here we report a turnkey microscopy system that enables simultaneous Ca 2+ 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. 

We have tested the feasibility of thermal grills, a harmless method to induce pain. The thermal grills consist of interlaced tubes that are set at cool or warm temperatures, creating a painful “illusion” (no tissue injury is caused) in the brain when the cool and warm stimuli are presented collectively. Advancement in objective pain assessment research is limited because the gold standard, the self-reporting pain scale, is highly subjective and only works for alert and cooperative patients. However, the main difficulty for pain studies is the potential harm caused to participants. We have recruited 23 subjects in whom we induced electric pulses and thermal grill (TG) stimulation. The TG effectively induced three different levels of pain, as evidenced by the visual analog scale (VAS) provided by the subjects after each stimulus. Furthermore, objective physiological measurements based on electrodermal activity showed a significant increase in levels as stimulation level increased. We found that VAS was highly correlated with the TG stimulation level. The TG stimulation safely elicited pain levels up to 9 out of 10. The TG stimulation allows for extending studies of pain to ranges of pain in which other stimuli are harmful.