Figure 1

Electrophysiological characterization of TRPV1-ChR2-EYFP transgenic mice and validation of the lick behavior. (A) (Top) Transgenic mice expressing ChR2 in TRPV1-containing neurons were bred using a Cre driver line. (Bottom) Immunohistochemistry of spinal cord tissue in TRPV1-ChR2-EYFP mice shows overlap of ChR2 (green) with CGRP (magenta), a marker of peptidergic nociceptors; scale bar = 150 µm. (B) Schematic of electrophysiological recording in anesthetized mice (left). Extracellular compound action potentials (CAP) were recorded from thoraco-lumbar vertebral levels evoked by 10 ms, 470 nm LED pulses applied to the ipsilateral hind paw of TRPV1-ChR2-EYFP mice. The CAP waveform average (right, n = 6 mice, mean ± S.E.M.) shows two prominent post-stimulus peaks corresponding to A-delta (t = 20 ms) and C-fiber (t = 200 ms) afferents. (C) Schematic layout of behavioral setup. Mouse is head-restrained with access to a lick spout, with their right hind paw restrained over a grated floor. Stimuli are delivered through the floor to the right hind paw. (D) Mice receive either optogenetic (target) or tactile (catch) stimuli. Following optogenetic stimulation, licking within the 1 s post-stimulus response window was considered a ‘hit’, while the absence of licking was considered a ‘miss’. Following tactile stimulation, licking within the 1 s response window was considered a ‘false alarm’, and the absence of licking was considered a ‘correct rejection’. (E) Example of a lick raster and histogram (left, n = 1 session), grey shading indicates the 1 s response window, ‘hit’ responses (green) occur during the 1 s response window, with only a small percentage (5%) of ‘miss’ trials (red). False alarm responses (yellow) occurred when the mouse responded in the 1 s response window following tactile stimuli. Psychometric response (right) for lick behavior fitted with a cumulative Gaussian function, compared to false alarm (FA) rate (dotted line). (F) Images captured showing representative reflex behavior (left) during two different trials that elicit a response (left, shaded green) vs. no response to optogenetic stimulation (right, shaded red) as compared to baseline pre-stimulus position (dotted lines). Psychometric response (right) for reflex behavior fitted with a cumulative Gaussian function, compared to FA rate (dotted line). All psychometric responses (E,F) showing mean ± SEM, n = 40 sessions from 5 mice. Data was plotted using MATLAB R2019a (https://www.mathworks.com). The figure was created using Adobe Illustrator CC 2019 (https://www.adobe.com).