Stimulus Generalization in Mice during Pavlovian Eyeblink Conditioning

Mouse ABRs and auditory eyelid startle responses to a range of sound frequencies and intensities. A, ABRs were measured at 4, 8, 16, and 32 kHz. Mice were most sensitive to sounds in the range of 8 and 16 kHz showing normal auditory thresholds. Each symbol represents one mouse. For the boxplot, the thick horizontal line is showing the median, the top edge of each box indicates the 25th percentile, bottom edge the 75th percentile, whisker lines extending above and below each box indicate the range of observations. B, The SPLs that elicits minimal auditory eyelid startle response for different sound frequencies. This value was determined for each mouse carefully during 10 baseline sessions and used as CS or GS.

Mouse eyelid startle responses obscure CR onset. A, Example eyelid responses to a 10-kHz tone in a naive unconditioned mouse presented at three different sound intensities: 60, 70, and 80 decibels (dB). In this example the 60-dB tone elicits just an α startle response, the 70-dB tone an α and β startle response, and the 80 an α and β startle and even a response that resembles a CR. For this mouse, a tone with a SPL of 60 dB would be a proper CS for training. B, Example eyelid responses after training taken from the same animal. In the left panel, the α startle response obscures the CR onset. In the right panel, there is no startle response and consequently the latency to CR onset can be detected reliably. C, Separation of startle and nonstartle trials was achieved by taking the first derivative of the eyelid position signal. In this velocity signal, the presence of a peak immediately after CS onset was the discriminator between startle (top panels) and nonstartle trials (bottom panels). Latency to CR onset was determined in nonstartle trials. For all other outcome measures, startle and nonstartle trials were combined. Similar to Figure 2, the blue gradient indicates GSs with frequencies higher than the 10-kHz CS and red gradient indicates GSs with frequencies lower than the 10-kHz CS. Each line is the averaged velocity signal of the eyeblink trace for one GS or CS frequency. For all panels, the green dashed line indicates CS onset, the red dashed line indicates expected US onset. The light green and red shadings indicate CS and US duration, respectively. Eyelid is fully open at 0 and fully closed at 1. The US is omitted in the CS-only trials. CS (tone), US (eye puff). D, Eyelid responses separated by the presence of an α startle response. Left panel shows eyelid traces with a startle response, right panel shows traces without a startle response.

Mice acquire conditioned eyelid responses over the course of 10 consecutive training sessions. A, Averaged eyeblink traces in CS-only trials during acquisition sessions 1, 4, 7, and 10 for the 11 mice that learned the task. The green dashed line indicates CS onset, the red dashed line indicates expected US onset. The light green and red shadings indicate CS and US duration, respectively. Eyelid is fully open at 0 and fully closed at 1. The US is omitted in the CS-only trials. B, CR percentage as a function of acquisition training session. Each solid gray line represents a mouse that did learn the task (n =11), each dotted gray line represents an animal that did not learn the task, i.e., did not reach a CR percentage of >20 after 10 training days (n = 3). Black line with black filled dots indicates the mean of each session for the 11 animals that learn the task. C, Eyelid closure amplitude over all trials plotted as a function of acquisition training session. The effect of session is statistically significant. For the boxplot, the thick horizontal line is showing the median, the top edge of each box indicates the 25th percentile, bottom edge the 75th percentile, whisker lines extending above and below each box indicate the range of observations, the plus symbols indicate outliers. The black line plot with filled black dots indicates the mean for each acquisition session. D, Distribution of eyelid closure amplitude calculated over all trials (acquisition sessions 8–10 pooled). Center of mass is around 0. For calculating the Eye closure – CR only trials, in panels in E–J, we used a CR criterium of 0.05 indicated with the vertical dashed line. E, Similar to C but now showing eyelid closure amplitude over CR only trials plotted as a function of acquisition training session. The effect of session is statistically significant. F, Similar to D, but now showing the distribution of eyelid closure amplitude calculated over CR only trials (acquisition sessions 8–10 pooled). G, Latency to CR peak plotted as a function of training session. The green dashed line indicates CS onset, the red dashed line indicates US onset. The light green and red shadings indicate CS and US duration, respectively. There is no statistically significant effect of session. H, Distribution of latency to CR peak for all trials (acquisition sessions 1–10 pooled). Note the adaptive timing of eyeblink CRs, whereby the CR peaks around the expected US (US is omitted in CS-only trials). I, Similar to G, but now showing latency to CR onset plotted as a function of training session. There is no statistically significant effect of session. J, Similar to H, but now showing the distribution of latency to CR onset for all sessions. For complete statistics for all panels, we refer to Table 1.

Generalization of conditioned eyelid responses in mice. A, CR percentage as a function of generalization test session. Solid gray lines represent individual mice and the black line with black filled dots indicates the mean of each session. No effect was found for generalization session, indicating that there was no extinction of eyeblink CRs during the generalization test sessions. B, CR percentage as a function of sound frequency. The 10-kHz tone is the CS, all other tone frequencies serve as GSs that are never reinforced with an air puff US. Solid gray lines represent individual mice, black line with black filled dots indicates the mean percentage CR for each GS. C, Eyelid closure amplitude over all trials plotted as a function of sound frequency. For the boxplot, the thick horizontal line is showing the median, the top edge of each box indicates the 25th percentile, bottom edge the 75th percentile, whisker lines extending above and below each box indicate the range of observations, the plus symbols indicate outliers. The black line plot with filled black dots indicates the mean for each sound frequency used. For panels C–H, the blue gradient indicates GSs with frequencies higher than the 10-kHz CS and red gradient indicates GSs with frequencies lower than the 10-kHz CS. D, Averaged eyeblink traces in response to different sound frequencies. The green dashed line indicates CS onset, the red dashed line indicates expected US onset. The light green and red shadings indicate CS and US duration, respectively. Eyelid is fully open at 0 and fully closed at 1. The US is omitted in the CS-only trials. Note the symmetric generalization gradient. E, Cumulative distribution function of eyelid closure calculated over all trials for the different sound frequencies. F, Similar to C, but now only for trials with a CR. G, Similar to D, but now for trials with a CR. H, Similar to E, but now for trials with a CR. I, Effect of sound frequency on the latency to CR peak. Lower tones tend to elicit eyeblink CR that peak earlier than higher tones. J, There was no effect of sound frequency on the latency to CR onset. For complete statistics, we refer to Tables 2-Tables 3.

Heatmaps showing adjusted p-values of all tone-tone comparisons for CR percentage, CR amplitude, and CR timing. A, Effect of tone frequency on CR percentage. The angular forked black box highlights the comparison between the 10-kHz CS and all the GSs. Note that the heatmap is on a logarithmic scale. All p-values were adjusted for multiple comparisons using FDR. Values correspond with those of Figure 5B. B, Effect of tone frequency on eyelid closure calculated over all trials. Values correspond with those of Figure 5C. C, Effect of tone frequency on eyelid closure calculated over CR only trials using a 0.05 criterium. Values correspond with those of Figure 5F. D, Effect of tone frequency on latency to CR peak. Values correspond with those of Figure 5I. E, Effect of tone frequency on latency to CR onset. Values correspond with those of Figure 5J. For complete statistics, we refer to Tables 2, 3.

Heatmaps showing adjusted p-values of all tone-tone comparisons for cumulative CR amplitude. A, Effect of tone frequency on cumulative CR amplitude calculated over all trials. Color indicates p-value. The angular forked black box highlights the comparison between the 10-kHz CS and all GSs. Note that the heatmap is on a logarithmic scale. All p-values were calculated using a Kolmogorov–Smirnov test on the cumulative distribution function (CDF). All p-values were adjusted for multiple comparisons using FDR. Values correspond with those of Figure 5E. B, Similar to A but now for the effect of tone frequency on cumulative CR amplitude calculated over CR only trials using 0.05 criterium. Values correspond with those of Figure 5H. For complete statistics, we refer to Table 4.

Heatmaps showing adjusted p-values of all tone-tone comparisons for cumulative CR amplitude using different CR thresholds. A, Effect of sound frequency on cumulative CR amplitude calculated over all trials. Color indicates p-value. The angular forked black box highlights the comparison between the 10-kHz CS and all GSs. Note that the heatmap is on a logarithmic scale. All p-values were calculated using a Kolmogorov–Smirnov test on the cumulative distribution function (CDF). All p-values were adjusted for multiple comparisons using FDR. Similar to Figure 5, the blue gradient indicates GSs with frequencies higher than the 10-kHz CS and red gradient indicates GSs with frequencies lower than the 10-kHz CS. B–D, Similar to A but now using a CR criteriums of 0.10, 0.15, and 0.20 FEC. E, Similar to B–D but now using the lowest CR threshold whereby there is a nonsignificant effect of GS for all frequencies. In mice, this threshold appeared to be 0.45. Thus, a threshold of 0.45 FEC was needed to get a binary response pattern, as reported previously (Khilkevich et al., 2018), whereby the probability of a CR gradually decreases depending on the similarity between CS and GS, but the amplitude of the CR remained constant. Note that this 0.45 is close to the split of the bimodal distributions shown in Figure 4D,F.