Apparatus and equipment configuration used for trace eyeblink conditioning in head-fixed mice (A), analysis of high-speed video (B), and example behavioral sessions from two mice (C). Apparatus and equipment configuration used for trace eyeblink conditioning in head-fixed mice (A), analysis of high-speed video (B), and example behavioral sessions from two mice (C). Mice did not show freezing (D) or startle responses (E) to presentation of a blue light CS. A, Mice were surgically implanted with a headplate to allow for fixation on a running wheel during training. CS included a blue LED CS and an air puff US. Eyelid behavior was monitored during trials with an infrared sensitive high-speed camera (200–250 fps). The black fur around the eye was illuminated with infrared light to contrast with a mouse’s eye and allow for analysis (see below). B, Standard trace conditioning trials consisted of a 200 ms baseline, 50 ms blue light CS, and a 250 ms stimulus-free trace interval followed by a 20 ms air puff US (top). Each frame of a trial was analyzed by calculating the ratio of white–black pixels within a specified region of interest (green rectangle in example sample frames; dashed lines show borders of white and black thresholded pixels), yielding the FEC at each sampled time point during a trial (right graphs; upward deflection indicates closure, red dashed line shows the minimum FEC to qualify as a CR, 0.1). Initially the mouse closed his eye only in response to the air puff (top), but with continued training learned to close his eye in response to the light CS and in anticipation of the air puff (bottom, CR). C, Waterfall plots show each trial of a session from two well trained mice (blue indicates presentation of light CS, gray indicates presentation of air puff US). Training sessions consisted of 60 trials, with CS-only probe trials presented every fifth trial (arrows) to allow for analysis of CRs in the absence of reflexive US responses. Note the upward deflection of the black lines prior to the air puff presentation for many of the trials, indicative of a CR. Bottom graphs show probe trials from the example sessions, which were used to calculate the latencies and amplitude of CRs for each mouse/session (see text). Note the absence of apparent startle responses just after CS presentation and the ramping topography of eyelid closures such that the peaks of CRs were well timed to US presentation (which were not presented during probe trials). D, Example wheel-running behavior from one mouse during its first trace eyeblink conditioning session (pseudocolor plot, each row is one trial, “+” indicates trials in which mouse was running before CS onset and included in analysis), and the averaged momentary speed before and after CS presentation (middle graph, median ± IQR). Bottom graph shows the averaged momentary speed for six mice (black lines), indicating that mice did not decrease locomotion (freeze) in response to the light CS (paired t = −1.31, df = 5, p = 0.25, n.s.). E. Example eyelid responses from four mice subjected to a startle-eliciting tone (top and bottom rows) or blue light CS (middle row), sampling at either 200 or 1500 fps as indicated. Startle responses to the tone were clearly detected when sampling at 200 fps, and were never observed in response to the light CS, even when sampling at 1500 fps. Jennifer J. Siegel et al. eneuro 2015;2:ENEURO.0051-14.2015 ©2015 by Society for Neuroscience