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. 2021 Jun 30;14:104. doi: 10.1186/s13041-021-00818-2

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Conditioning-induced activation of orexin neuron and increase in heart rate without aversive sensory input. A Experimental design for neuronal and heart rate recordings of conditioning group (n = 4). Heart rate (HR) and fluorescence via fiber photometry (FP) were recorded for 2 min around sound application before conditioning (pre) and 1 h after conditioning (post). HR and fluorescence were averaged for 1 min during the baseline period and after the cue sound (green horizontal line). B Traces of the heart rate (upper), G-CaMP6 and mCherry fluorescence (lower) in pre-conditioning (left) and post-conditioning (right) in the conditioning group. The dark-colored line indicates the averaged value and the light-colored line shows SEM. C Grouped average and SEM of the averaged values for 1 min during the baseline period and after the cue sound. Two-way ANOVA revealed that there was significant difference between baseline and cue sound (Heart rate: F(1, 3) = 8.719, P = 0.0599; G-CaMP6: F(1,3) = 15.22, P = 0.0299; mCherry: F(1,3) = 2.096, P = 0.2435) and between pre-condition and post-condition (Heart rate: F(1, 3) = 622.9, P < 0.0001; G-CaMP6: F(1,3) = 30.91, P = 0.0115; mCherry: F(1,3) = 0.04808, P = 0.8405). P values in the figure were calculated by Bonferroni’s multiple comparison test. D Changes in the rate of increase due to the cue sound of individual mice. The rate of increase is represented by the average of after cue sound/the average of before cue sound. P values were calculated using a paired t-test. E The difference between the onset of fluorescence change and the onset of heart rate change. See Fig. 3A for the definition of ∆time. F Experimental design for neuronal and heart rate recordings in the control group (n = 11). G Traces of the heart rate (left), G-CaMP6 and mCherry fluorescence (right) in the control group. H Grouped average and SEM of the averaged values for 1 min during the baseline period and after the cue sound. P-values were calculated using paired t-test. I Results of the freezing score (% time) during cue test tone. In the post-session, freezing behavior was counted in the control group (n = 11) and in the conditioned group (n = 4). P-values were calculated using Mann–Whitney’s nonparametric test

Fig. 4 — Conditioning-induced activation of orexin neuron and increase in heart rate without aversive sensory input. A Experimental design for neuronal and heart rate recordings of conditioning group (n = 4). Heart rate (HR) and fluorescence via fiber photometry (FP) were recorded for 2 min around sound application before conditioning (pre) and 1 h after conditioning (post). HR and fluorescence were averaged for 1 min during the baseline period and after the cue sound (green horizontal line). B Traces of the heart rate (upper), G-CaMP6 and mCherry fluorescence (lower) in pre-conditioning (left) and post-conditioning (right) in the conditioning group. The dark-colored line indicates the averaged value and the light-colored line shows SEM. C Grouped average and SEM of the averaged values for 1 min during the baseline period and after the cue sound. Two-way ANOVA revealed that there was significant difference between baseline and cue sound (Heart rate: F(1, 3) = 8.719, P = 0.0599; G-CaMP6: F(1,3) = 15.22, P = 0.0299; mCherry: F(1,3) = 2.096, P = 0.2435) and between pre-condition and post-condition (Heart rate: F(1, 3) = 622.9, P < 0.0001; G-CaMP6: F(1,3) = 30.91, P = 0.0115; mCherry: F(1,3) = 0.04808, P = 0.8405). P values in the figure were calculated by Bonferroni’s multiple comparison test. D Changes in the rate of increase due to the cue sound of individual mice. The rate of increase is represented by the average of after cue sound/the average of before cue sound. P values were calculated using a paired t-test. E The difference between the onset of fluorescence change and the onset of heart rate change. See Fig. 3A for the definition of ∆time. F Experimental design for neuronal and heart rate recordings in the control group (n = 11). G Traces of the heart rate (left), G-CaMP6 and mCherry fluorescence (right) in the control group. H Grouped average and SEM of the averaged values for 1 min during the baseline period and after the cue sound. P-values were calculated using paired t-test. I Results of the freezing score (% time) during cue test tone. In the post-session, freezing behavior was counted in the control group (n = 11) and in the conditioned group (n = 4). P-values were calculated using Mann–Whitney’s nonparametric test