Article

Locomotor activity modulates associative learning in mouse cerebellum

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Abstract

Changes in behavioral state can profoundly influence brain function. Here we show that behavioral state modulates performance in delay eyeblink conditioning, a cerebellum-dependent form of associative learning. Increased locomotor speed in head-fixed mice drove earlier onset of learning and trial-by-trial enhancement of learned responses that were dissociable from changes in arousal and independent of sensory modality. Eyelid responses evoked by optogenetic stimulation of mossy fiber inputs to the cerebellum, but not at sites downstream, were positively modulated by ongoing locomotion. Substituting prolonged, low-intensity optogenetic mossy fiber stimulation for locomotion was sufficient to enhance conditioned responses. Our results suggest that locomotor activity modulates delay eyeblink conditioning through increased activation of the mossy fiber pathway within the cerebellum. Taken together, these results provide evidence for a novel role for behavioral state modulation in associative learning and suggest a potential mechanism through which engaging in movement can improve an individual’s ability to learn.

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Acknowledgements

We thank T. Pritchett for technical assistance and P. Francisco for help on some of the auditory CS experiments. We thank G. Costa for illustrations and the Champalimaud Research Hardware Platform for technical support. We thank J. Fayad and M. Orger for advice on data analysis. We are grateful to the Carey lab and the members of the Champalimaud Neuroscience Program for helpful discussions throughout the project and in particular to H. Marques and J. Jacobs for comments on the manuscript. This work was supported by a Howard Hughes Medical Institute International Early Career Scientist Grant #55007413 (to M.R.C.), Bial Foundation Bursary #74/14 (to D.L.P.), fellowships from the Portuguese Fundação para a Ciência e a Tecnologia #BD77686/2011 (to C.A.) and #BPD109659/2015 (to D.L.P.), and European Research Council Starting Grant #640093 (to M.R.C.).

Author information

Affiliations

  1. Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Lisbon, Portugal

    • Catarina Albergaria
    • , N. Tatiana Silva
    • , Dominique L. Pritchett
    •  & Megan R. Carey

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  1. Search for Catarina Albergaria in:

  2. Search for N. Tatiana Silva in:

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Contributions

C.A. and M.R.C. designed the research plan. C.A. and N.T.S. performed all experiments. C.A. and D.L.P. performed electrophysiological recordings. D.L.P. analyzed electrophysiology data. C.A. analyzed all data and prepared figures. C.A. and M.R.C. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Megan R. Carey.

Integrated supplementary information

  1. Supplementary Figure 1 Rate and extent of learning depend on running speed.

    A. Amplitude acquisition curve for one representative animal (gray line) fitted with a sigmoid (gray line). B. Slope of sigmoid fits of the acquisition curve for each animal. The magenta line is a linear fit (N=28, slope = 4.515, **p = 0.0017). C. Plateau of sigmoid fit for each animal. The magenta line is a linear fit (N=28, slope = 0.8, *p = 0.0376). D. As above, for fast (magenta) and slow (cyan) motorized treadmill. E. Slope of sigmoid fits of the acquisition curve for animals from each speed group superimposed on the self-paced treadmill data (grey) from (B). F. Plateau of sigmoid fit for animals from each speed group superimposed on the self-paced treadmill data (grey) from (C).

  2. Supplementary Figure 2 Influence of arousal vs. locomotor activity on CR amplitude.

    A. Relationship between pupil size and walking speed for all trials from all training sessions using a whisker (in yellow) or a tone (in red) CS. Line is average across animals; shadow indicates SEM. There was a significant positive relationship for both whisker (mixed ANOVA, n=40307 trials, N=25 animals, F(1,214.5) = 140.75, ***p = 4.82e-24) and tone (n=27204 trials, N=16 animals, F(1,130) = 127.3, ***p = 5.24e-21). B. Relationship between CR amplitude and pupil size for all CR's to a whisker or a tone CS. Line is average across animals; shadow indicates SEM. No correlation was found for either whisker (one-way ANOVA on LME, F(1,225) = 0.203, p = 0.6524) or tone (one-way ANOVA on LME, F(1,162) = 0.1736, p = 0.6775) CS. C,D. Analyzing the effects of locomotor activity separately for trials with high arousal (pupil > 0.85) and low arousal (pupil < 0.85) for a whisker CS (C, mixed ANOVA, high arousal: n=6277 trials, N=25 animals, F(1,154.9) =8.64, p < 0.01; low arousal: n=9291 trials, N=25 animals, F(1,105.8) = 7.03, **p < 0.01) and a tone CS (D, mixed ANOVA, high arousal: n=3462 trials, N=16 animals, F(1,76.6) = 22.5, ***p < 0.001; low arousal: n=6089 trials, N=16 animals, F(1,66) = 42.75, ***p < 0.001). E,F. Analyzing the effects of arousal separately for trials with higher (speed > 0.15m/s) and lower walking speed (speed < 0.15m/s) for a whisker CS (E, mixed ANOVA, high speed: n=3781 trials, N=25 animals, F(1,170.9) = 1.66, p = 0.20; low speed: n=12003 trials, N=25 animals, F(1,269.1) = 20.25, ***p < 0.001) and a tone CS (F, mixed ANOVA, high speed: n=1545 trials, N=16 animals, F(1,112.35) = 0.21, p = 0.65; low speed: n=8276 trials, N=16 animals, F(1,155.63) = 9.30, **p < 0.01). Histograms under each plot represent the number of animals in each bin for each group. G. Analyzing the effects of arousal (as measured by pupil size) for trials when the animals were still, on CR's evoked by a whisker (green) or a tone (red) CS. Line is average across animals; shadow indicates SEM. There was a significant negative relationship between pupil size and CR amplitude for both whisker (mixed ANOVA, n=9223 trials, N=25 animals, F(1,257.8) = 35.4, ***p < 0.0001) and tone (n=6930 trials, N=16 animals, F(1,191.3) = 22.4, ***p < 0.0001).

  3. Supplementary Figure 3 Fiber optic placement for optogenetic experiments.

    Representative histological samples indicating fiber placements for the optogenetic manipulations described in Figs. 4, 5, and 6. Green fluorescence indicates YFP (ChR2) expression. Dashed white circles indicate the site where the DiI coated optic fiber was implanted. Bottom two rows: Colored circles represent sites of implanted optical fibers from additional animals. A. MF-ChR2-ctx: Optical fibers were placed in an eyelid region of the cerebellar cortex of Thy1-ChR2-YFP mice expressing ChR2:YFP in a subset of mossy fibers. B. gc-ChR2-ctx: Optical fibers were placed in an eyelid region of the cerebellar cortex of Gabra6cre-ChR2-YFP mice in which ChR2:YFP was targeted to cerebellar granule cells. C. Pkj-ChR2-ctx: Optical fibers were placed in an eyelid region of the cerebellar cortex of L7cre-ChR2-YFP mice expressing ChR2:YFP in Purkinje cells. D. MF-ChR2-AIP: Optical fibers were placed in the anterior interpositus nucleus of Thy1-ChR2-YFP mice. IntA, interposed cerebellar nucleus, anterior part. Lat, lateral cerebellar nucleus. Med, medial cerebellar nucleus.

  4. Supplementary Figure 4 Cell-type-specific ChR2-YFP expression.

    Images at different magnifications from MF-ChR2-YFP, gc-ChR2-YFP and Pkj-ChR2-YFP lines demonstrating cell-type specific YFP/ChR2 expression in each line. Green fluorescence indicates YFP (ChR2) expression. A. Fluorescence images of parasagittal cerebellar sections of a Thy1-ChR2-YFP mouse expressing ChR2:YFP in a subset of mossy fibers and their terminals. Center, specific expression in MF terminals within the cerebellar cortex. Right, MF terminals labeled within the AIP. B. Fluorescence images of parasagittal cerebellar sections of a Gabra6Cre-ChR2-YFP mouse indicating ChR2:YFP expression selectively in granule cells (gc-ChR2). C. Fluorescence images of parasagittal cerebellar sections of a L7Cre-ChR2-YFP mouse expressing ChR2:YFP selectively in Purkinje cells.

  5. Supplementary Figure 5 Photostimulation controls.

    A. gc-ChR2-AIP: Fluorescence image of a coronal cerebellar slice of a Gabra6cre-ChR2-YFP mouse expressing ChR2:YFP in cerebellar granule cells. The Dil coated optical fibers were placed in the anterior interpositus nucleus (AIP). IntA, interposed cerebellar nucleus, anterior part. Lat, lateral cerebellar nucleus. Med, medial cerebellar nucleus. B. Eyelid movements in response to moderate to high intensity laser stimulation (473nm light pulses at 100Hz for 50ms) in Gabra6cre-ChR2-YFP mice expressing ChR2:YFP in cerebellar granule cells. Laser-driven responses from mice with the fiber implanted in the anterior interpositus nucleus (gray, N=5) are shown. For comparison, data from the same mouse line, but the fiber implanted in cerebellar cortex, is replotted (green) from Fig. 5E. Duration of laser stimulation is indicated by the horizontal black line. C. Eyelid responses to high intensity laser stimulation (473nm light pulses at 100Hz for 50ms) in wild-type mice (N=4, all superimposed) with an optic fiber implanted in an identified eyelid-related region of cerebellar cortex.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–5

  2. Reporting Summary

  3. Supplementary Video 1

    Head-fixed mouse running on a motorized treadmill during an eyeblink conditioning session with a visual CS and an airpuff US.