Locomotor activity modulates associative learning in mouse cerebellum

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.

Fig. 1. Eyeblink conditioning performance correlates with locomotor activity across mice, sessions, and trials

A. Setup schematic for eyeblink conditioning in head-fixed mice on a running wheel, illustrating a white LED as the CS and an air puff US. B. Average eyelid closure for a representative animal across 9 learning sessions (S1-S9). Each trace represents the average of 100 paired trials from a single session. Example video frames (acquired at 900 fps under infrared light) illustrate automated extraction of eyelid movement amplitude. C. Cumulative histogram of locomotor activity of all animals (N=34) trained with a light CS, calculated by averaging the average speed of each session. D. Learning curves for all animals represented in (C), color-coded for their average speed. Averages of the 20% fastest and slowest mice are superimposed in magenta and cyan, respectively. E. Onset session of learning for each animal, plotted against the animals’ average walking speed. Onset session was defined as the session in which the average CR amplitude exceeded 0.1. Each dot represents an animal. The line is a linear fit (N=34, slope = -34.4, ***p = 0.00057). See also Supp. Fig. 1. F. Session-to-session changes in average CR amplitude (y-axis) are plotted as a function of session-to-session changes in locomotor activity (x-axis), for all learning sessions, color-coded for the average speed of each session. There was a significant positive relationship (one-way ANOVA on linear mixed effects model (LME), n=646 sessions, N=34 animals, F(1,155) = 24.271, ***p = 2.127e-6) between changes in walking speed and changes in CR amplitude. G. Average of trials from session 6 of one representative animal, divided into 3 speed intervals (<0.1m/s (18 trials); 0.2-0.25m/s (20 trials); >0.35m/s (16 trials)) and color-coded accordingly. Shadows represent SEM. H. Trial-to-trial correlation between CR amplitude and walking speed. CR amplitudes for all trials from the session in which each individual animal crossed a threshold of 50% CR plus the following session are plotted. Line is average across animals; shadow indicates SEM. There was a linear positive relationship (one-way ANOVA on LME, n=7,480 trials, N=34 animals, F(1,180.29) = 83.023, ***p = 1.55e-16) between running speed and conditioned response amplitude. I. Correlation between unconditioned response (UR) magnitude and walking speed from trial-to-trial. The normalized area under the UR in response to the air puff is plotted for Session 1, before emergence of conditioned responses. Line is average across animals; shadow indicates SEM. There was a linear negative relationship (one-way ANOVA on LME, n=3,332 trials, N=34 animals, F(1,91.898) = 27.366, ***p 1.05e-6) between running speed and UR amplitudes.

Fig. 2. Speed-dependent modulation of eyeblink conditioning on a motorized treadmill

A. Individual learning curves with superimposed averages of two groups of mice, running on either a faster (magenta, 0.18m/s, N=7) or slower (cyan, 0.12m/s, N=5) motorized treadmill. B. Quantification of learning onset session for each animal. Fast (magenta) and slow (cyan) motorized data are superimposed on the self-paced treadmill data from Fig. 1E (gray). The difference in learning onset between the fast and slow group was significant (average session 3.4 vs. 6.4, respectively, ***p = 2.79e-6, Student’s two-sided t-test). C. Eyelid traces of individual trials for individual representative animals on the fast (magenta) vs. slow (cyan) motorized treadmills at learning session 7. The traces for every 10th trial are shown. D. Median CR amplitudes from S7 for animals (dots) running at the slow (s, cyan, N=5) or fast (f, magenta, N=7) motorized speed (fast vs. slow, **p = 0.003, Student’s two-sided t-test). Box indicates median and 25^th-75^th percentiles, whiskers extend to the most extreme data points. Significance: *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 3. Modulation of CR acquisition and amplitude are CS-independent and dissociable from effects of arousal

A. Schematic for experiments using conditioned stimuli of different sensory modalities: light CS in gray, tactile (whisker) CS in orange and auditory (tone) CS in red; each was paired with an airpuff US. B. Trial-to-trial correlation between CR amplitude and walking speed for all trials with CRs 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 (one-way ANOVA on LME, n=15490 trials, N=25 animals, F(1,159.8) = 11.499, ***p = 0.0009) and tone (n=9771 trials, N=16 animals, F(1,82.5) = 32.255, ***p = 1.968e-7). C. Onset learning session for animals from all three CS modalities, color-coded as in (A), plotted against the average walking speed of each animal on the self-paced treadmill. The lines are linear fits (visual CS from Fig. 1E; whisker CS: N=25 animals, slope=-15.7; p=0.052; tone CS: N=16 animals, slope=-8.9; p=0.49). D. Individual whisker CS learning curves with averages superimposed of two groups of mice running on either a faster (green, 0.18m/s, N=6) or slower (orange, 0.12m/s, N=5) motorized treadmill. E. Quantification of learning onset session for each animal from (D). Fast (green) and slow (orange) motorized data are superimposed on the self-paced treadmill (yellow). The difference in onset learning between the fast and slow group was significant (***p = 1.92e-4, Student’s two-sided t-test). F. Eyelid traces of individual trials for a representative animal on the fast (green) vs. slow (orange) motorized treadmill at S10. G. Quantification of CR amplitudes from S10 for animals (dots) running either at the slow (orange) or fast (green) motorized speed. The slow and fast groups were significantly different (**p = 0.0094, Student’s two-sided t-test). Box indicates median and 25^th-75^th percentiles, whiskers extend to the most extreme data points. H. Relationship between CR amplitude and pupil size for all CR trials from training sessions using a whisker CS on the fast (one-way ANOVA on LME, n=8249 trials, N=6 animals, F(1,102) = 10.35, **p = 0.0017) versus slow (one-way ANOVA on LME, n=4194 trials, N=5 animals, F(1,38) = 2.929, p = 0.095) motorized treadmill. Line is average across animals; shadow indicates SEM. Inset: Median pupil size (pixels) for both speeds (difference not significant, p = 0.5109, Student’s two-sided t-test). Box indicates median and 25^th-75^th percentiles, whiskers extend to the most extreme data points. Significance: *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 4. Conditioned responses acquired with optogenetic stimulation of cerebellar mossy fibers in the cerebellar cortex are positively modulated by locomotor activity

A. Cerebellar circuit diagram with a blue lightning bolt representing the site of laser stimulation: mossy fiber terminals in eyelid-related cerebellar cortex. B. Schematic of eyelid conditioning protocol using MF-ChR2-ctx optogenetic stimulation as a replacement for the CS. Animals implanted with optical fibers in an eyelid-related region of cerebellar cortex were trained to a sub-threshold (i.e. not eliciting eye movement) laser stimulation of mossy fibers (473nm light pulses at 100Hz for 350ms) paired with an airpuff to the eye as US. C. Representative individual trials for one mouse trained with a MF-ChR2-ctx CS and airpuff US, during the first eight sessions. The eyelid trace for every 9th trial from eight sessions is plotted. D. Average %CR learning curves to MF-ChR2-ctx CS’s for animals walking on a self-paced treadmill (filled circles, N=7), and animals running at a fast fixed speed (0.18m/s, N=7) on the motorized treadmill (open circles). To control for the possibility that the mice could see the laser, which could inadvertently act as a visual CS, wildtype controls (not expressing ChR2) were implanted with optical fibers and underwent the same training protocol (black circles, N=4). Error bars indicate SEM. E. Learning onset session for the animals in D are superimposed on the self-paced treadmill data from mice trained to a visual CS (gray). The blue line is a linear fit (onset value: slope = -16.8, **p = 0.00015). F G H After learning reached a plateau, both groups were tested for the expression of CRs in test sessions at two fixed speeds on the motorized treadmill: slow (0.06m/s) and fast (0.18m/s). (F) Average of CS-only trials (n=50 trials) from the slow (dashed line) and fast (solid line) blocks of trials, for one representative animal. Shadows indicate SEM. Vertical dashed lines represent the time that the US would have been expected on CS+US trials. (G) Average CR amplitude of responses from each animal (N=15) walking at slow (0.06m/s) vs fast (0.18m/s) pace on the motorized treadmill. The average from all animals is superimposed (in blue, **p = 0.0017, Student’s two-sided paired t-test). (H) Relationship between CR amplitude and pupil size for all CR trials from test sessions with an optogenetic CS on the fast (solid line, one-way ANOVA on LME, n=1501 trials, N=15 animals, F(1,55.3) = 0.845, p = 0.36192) vs. slow (dashed line, one-way ANOVA on LME, n=1501 trials, N=15 animals, F(1,11.9) = 59.5, *p = 0.001) motorized treadmill. Line is average across animals; shadow indicates SEM. Significance: *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 5. Eyelid closures evoked by optogenetic MF stimulation in the cerebellar cortex are positively modulated by locomotion

A. Schematic of the cerebellar circuit including some of the major cell types in the cerebellar cortex and deep nuclei. Lightning bolts represent the different targets of laser stimulation: mossy fibers (MF) terminals in the cerebellar cortex (blue), MF terminals in the deep nuclei (cyan), granule cells (gc, green) and Purkinje cells (Pkj, pink). IN, interneuron; PF, parallel fiber; AIP, anterior interpositus. B-E. Eyelid movements from a representative animal from each mouse line in response to supra-threshold laser stimulation (473nm light pulses at 100Hz for 50ms) at two different intensities. Vertical dashed lines represent the stimulus duration; average (of 10-20 trials each) represented by the thick line; shadows indicate SEM. Laser pulse duration is indicated by the vertical dashed lines. B,C. An optical fiber was placed in an identified eyelid-related region of cerebellar cortex (B, blue) or AIP (C, cyan) of Thy1-ChR2-YFP mice that express ChR2 in cerebellar mossy fibers. D,E. An optical fiber was placed in the same eyelid region of cerebellar cortex in mice that express ChR2 in Purkinje cells (L7cre-ChR2-YFP, D, pink) or cerebellar granule cells (Gabra6cre-ChR2-YFP, E, green). F-I. In vivo electrophysiological responses to 50 ms laser stimulation in awake mice for the lines depicted in (B-E). Example extracellular traces are shown above the peri-stimulus time histograms and laser pulse durations are indicated by the shadows of corresponding colors. F. In vivo recordings from units in cerebellar cortex in response to MF-ChR2-ctx stimulation. G. In vivo recordings from units in cerebellar nuclei in response to MF-ChR2-AIP stimulation. H. In vivo recordings from units in cerebellar cortex in response to Pkj-ChR2-ctx stimulation. I. In vivo recordings from units in cerebellar cortex in response to gc-ChR2-ctx stimulation. J. Correlation between laser-driven eyelid responses and walking speed on the self-paced treadmill from trial-to-trial, for MF-ChR2-ctx mice (blue, n=1072 trials, N=8 animals; one-way ANOVA on LME, F(1,1068.8) = 5.01, *p = 0.025); MF-ChR2-AIP mice (cyan, n=502 trials, N=5 animals; F(1,501.8) = 4.02, *p = 0.04); and gc-ChR2-ctx mice (green, n=943 trials, N=11 animals; F(1,939.5) = 30.68, ***p = 3.95e-08). Lines represent trial-wise averages from all animals and error bars indicate SEM. K-N. Average amplitude of laser-elicited eye closures from animals walking at slow (0.06m/s) or fast (0.18m/s) pace, as set by the motorized treadmill. Each animal was tested for the two speeds within one session, using the same stimulation protocol and laser intensity. The average from all animals is superimposed. K. Laser-elicited blink in MF-ChR2-ctx mice (N=7; *p = 0.0298, Student’s paired t-test); L. laser-elicited blink in MF-ChR2-AIP mice (N=6; *p = 0.0197, Student’s two-sided paired t-test); M. Laser-elicited blink in Pkj-ChR2-ctx mice (N=10; p = 0.9361, Student’s two-sided paired t-test); N. Laser-elicited blink in gc-ChR2-ctx mice (N=11; *p = 0.03, Student’s two-sided paired t-test). Significance: *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 6. Low-level background mossy fiber stimulation is sufficient to enhance conditioned response amplitude

A. Schematic illustrating proposed mechanism for CR modulation by locomotor activity. Distinct mossy fiber terminals (depicted here as one CS mossy fiber and one ‘locomotor’ mossy fiber) converge onto individual granule cells. Summation of multiple mossy fiber inputs is required for postsynaptic granule cell firing. Enhanced mossy fiber tone during locomotion (bottom) therefore leads to enhanced granule cell CS responses relative to when the mouse is stationary (top). B. Experimental design: After training MF-ChR2-YFP mice implanted with optical fibers in an eyelid-related region of cerebellar cortex, using a visual CS and an airpuff US, trials were presented in alternating blocks of 10 trials, either without stimulation (‘no tickle’ block), or with extremely low intensity 50 Hz, 2ms pulses, background optogenetic stimulation of mossy fibers in the cerebellar cortex (‘tickle’ block). Motorized treadmill speed was fixed (0.12m/s) throughout the experiment. C. In vivo electrophysiological responses to 20 s laser stimulation (50 Hz, 2ms pulses) delivered through an optical fiber implanted in the eyelid region of cerebellar cortex in an awake MF-ChR2-YFP mouse. An example extracellular trace is shown above the peri-stimulus time histogram and laser pulse duration is indicated by the blue shadow. D. Average of eyelid traces from ‘tickle’ (blue) and ‘no tickle’ (gray) blocks (21 trials in each average), for a representative animal. Shadows indicate SEM. E. Comparison of CR amplitudes with (left) vs without (right) laser stimulation, in MF-ChR2 mice (blue, N=6, **p = 0.0031, Student’s two-sided paired t-test) and wildtype controls (not expressing ChR2, black, N=4, p = 0.6014, Student’s two-sided paired t-test). Thin lines represent individual animals, thick lines are averages across animals. Error bars represent SEM. Significance: *p < 0.05, **p < 0.01 and ***p < 0.001.