Cerebellar modulation of the reward circuitry and social behavior

Abstract

The cerebellum has been implicated in a number of nonmotor mental disorders such as autism spectrum disorder, schizophrenia, and addiction. However, its contribution to these disorders is not well understood. In mice, we found that the cerebellum sends direct excitatory projections to the ventral tegmental area (VTA), one of the brain regions that processes and encodes reward. Optogenetic activation of the cerebello-VTA projections was rewarding and, in a three-chamber social task, these projections were more active when the animal explored the social chamber. Intriguingly, activity in the cerebello-VTA pathway was required for the mice to show social preference in this task. Our data delineate a major, previously unappreciated role for the cerebellum in controlling the reward circuitry and social behavior.

Fig. 1.. Optogenetic activation of cerebellar axons in the VTA drives VTA activity in vivo

A, ChR2 was expressed in the DCN. An optrode was lowered to the VTA to simultaneously stimulate cerebellar axons in the VTA and record single-unit activity of VTA neurons. Example injection site shown on right. B, Example single unit recording from the VTA. The timing of the stimulus (1 ms, 2 mW) is indicated by the blue triangle. C,D, Example activity rasters and resulting firing rate histograms following repeated trials of single pulse optical stimulation of cerebellar axons in two neurons in the VTA. Stimulus was delivered at time zero. E, 36% of VTA cells responded to optogenetics activation of cerebellar axons in the VTA (n/N = 103/14). F, Latency histogram of VTA neurons excited by optogenetic activation of cerebellar axons in the VTA (mean latency: 5.9±0.5 ms, SEM; median: 6 ms). G, Example raster and firing rate histogram following a 20 Hz train of light pulses to optogenetically activate cerebellar axons in the VTA. Train started at time zero; each pulse is indicated by a blue triangle. H, Average response to 20 Hz trains in all VTA neurons examined (n/N = 14/3). Train onset at time zero; each pulse is indicated by a blue triangle. I, Average extra spikes elicited by a 20 Hz train (n/N = 14/3; Mean ± SEM).

Fig. 2.. Cerebellar axons in the VTA form monosynaptic glutamatergic synapses

A, ChR2 was expressed in the DCN. Whole cell recordings were made in the VTA (indicated in red). Blue light (447 nm) was delivered through the objective to stimulate cerebellar axons in the VTA. (HP: hippocampus, CC: corpus callosum, VTA: ventral tegmental area). B, Cells in the VTA fired action potentials in response to stimulation of cerebellar axons in cell-attached recordings. Blue triangle indicates timing of the 1 ms laser pulse. C, Optogenetic activation of cerebellar axons in the VTA resulted in EPSCs in the VTA neurons which were blocked by CNQX. Left: response of a VTA neuron clamped at −70 mV to stimulation of cerebellar axons before (black) and after (red) bath application of CNQX. Right: average decrease in response amplitude following application of CNQX. Each symbol represents a cell; data are represented as mean ± SEM (n = 9). (p = 0.002, Wilcoxon Signed Ranks). D, Optogenetically activated responses were monosynaptic. Optically evoked responses were blocked by bath application of 1 µM TTX. Responses could be recovered with subsequent application of 200 µM 4-AP. Example shown on left; summary data for cells recorded in aCSF (n = 24), TTX (n = 9), and 4-AP+TTX (n = 11). (ACSF vs. TTX+4AP: p = 0.4238, ACSF vs TTX: p<0.0001, TTX vs. TTX+4AP: p<0.0001. Wilcoxon Rank Sum) E, When the VTA neurons were clamped at +50 mV (blue), a second, slower decay time constant was observed in addition to the fast decay time constant seen at a holding potential of −70 mV (black, n = 10), which corresponded with the AMPA component. F, Currents observed at +50 mV are due to NMDA. NMDA currents were isolated using NBQX and blocked by AP5. Example currents at +50 mV shown on top; group data shown on bottom. Each symbol represents a cell, and data are represented as mean ± SEM (n=5). (p = 0.0313, Wilcoxon Signed Rank) G, Cerebellar inputs to the VTA show synaptic depression. Example 20 Hz stimulus trace shown on top. Average responses to 5, 10, and 20 Hz trains (n = 5, 6, 11 respectively). H, Cerebellar stimulation produces responses in both TH+ and TH- neurons in the VTA. Cells within the VTA were whole cell patch clamped with an internal solution containing neurobiotin, and post hoc stained for TH (n = 29). Two example cells (indicated by white arrows) are shown: one was co-stained with TH (right) while the other was not (left). Response percentages shown on the bottom. Proportion of responding TH+ cells was not significantly different from the proportion of TH- cells (p = 0.4636, Chi Square Test). I, Anterograde trans-synaptic tracing indicates that the cerebellum sends inputs to both TH+ and TH- neurons within the VTA. A GFP-tagged H129 strain of herpes simplex virus type 1 (H129-GFP) was injected into the DCN and incubated for 50 hours—sufficient time to cross only one synapse (Fig. S2).

Fig. 3.. Stimulation of cerebellar axons in the VTA is rewarding

A, Optogenetic stimulation protocol. A train of 1ms pulses at 20 Hz for 3 seconds was delivered repeatedly every 10s in a chosen quadrant of the test arena. B+J, ChR2 was expressed in the DCN and fiber-optics were bilaterally implanted targeting the VTA to allow optogenetic activation of cerebellar axons. C, Mice were tested in a behavioral assay by placing them in a square chamber and allowing them to explore it at will. After obtaining a baseline record for ten minutes (top row), one of the quadrants was subsequently chosen to be the reward quadrant and the behavior followed for another ten minutes (middle row). Upon entry to the reward quadrant, cerebellar axons in the VTA were optically stimulated as described in A. This stimulus was repeated every 10 seconds as long as the mouse stayed in the reward quadrant. Afterwards (bottom row), the reward quadrant was re-assigned to a different quadrant in the chamber and the experiment repeated. D+E, Mice expressing ChR2 in the Cb-VTA pathway exhibited a marked preference for the reward quadrant. A single trial example (D), and the average (E) of all mice during behavioral task outlined above. The reward quadrant is indicated by the white box. F+N, In a cohort of DAT-CRE mice, ChR2 was expressed in the VTA dopaminergic cells and fiber-optics were bilaterally implanted targeting the VTA. G+H, DAT-CRE mice expressing ChR2 in dopaminergic cells exhibited a preference for the reward quadrant,. A single trial example position map (G), and the average (H) of all mice during behavioral task I, Variation to optogenetic stimulation protocol in a: a train of 1ms pulses at 20 Hz for 3 seconds was delivered only upon entry in a chosen quadrant of the test arena. K, Behavioral paradigm as in c. However, the stimulus was delivered only upon entry to the chosen quadrant. To receive more stimulation, the mice are required to leave and re-enter the quadrant. L+M, Mice expressing ChR2 in the Cb-VTA pathway exhibited preference for the reward quadrant in the modified self-stimulation task. L, Single trial example. M, Average session across all mice tested. The reward quadrant is indicated by the white box. O+P, DAT-CRE mice expressing ChR2 in dopaminergic VTA cells exhibit preference for the reward quadrant in the modified version of the Self Stimulation task. O, Single trial example of mouse position map. P, Average session across all mice tested. The reward quadrant is indicated by the white box. Q, When stimulated with the protocol in A: Mice expressing ChR2 in the Cb-VTA pathway exhibited a strong preference for the reward quadrant. (N = 22, p<0.0001), DAT-CRE mice exhibited a similar preference (N=8, p<0.0001). When stimulated with protocol in I: both groups exhibited a strong preference for the reward quadrant: Cb-VTA (N=17; 16 with bilateral, 1 with unilateral implant, p<0.0001), DAT-CRE (N=8, p<0.0001). GFP expressing animals stimulated with protocol in A did not show a preference for any of the quadrants (N=12, p>0.9999). Stimulation Trials 1 and 2 were averaged. (Two way ANOVA followed by Bonferroni post-hoc test. Data are mean ± SD). R, After each stimulation trial, a subset of mice expressing ChR2 in the Cb-VTA pathway was examined for an additional 15 minutes without delivering additional laser stimulations. A residual preference for the last reward quadrant was noted only during the first minute (N=16, p<0.0001). Stimulation Trials 1 and 2 were averaged. (Two way ANOVA followed by Bonferroni post-hoc test. Data are mean ± SD).

Fig. 4.. Activation of cerebellar inputs to VTA promotes conditioned place preference.

A, ChR2 was expressed in the DCN and fiber-optics were bilaterally implanted targeting the VTA to allow optogenetic activation of cerebellar axons. B+C, Experimental paradigm. Mice were tested in a conditioned place preference (CPP) apparatus containing two chambers, differentiated by lighting conditions and walls of each chamber showing stripes of opposing orientations. On Day 1, animals were allowed to freely explore the apparatus for 15 minutes, to establish a baseline chamber preference. Beginning on Day 2, mice were conditioned for 30 minutes per day, on 4 consecutive days, for 3 weeks. Mice were alternately restricted to either the lighted or dark chamber. While confined to the lighted chamber, subjects received 3 s, 20 Hz trains of optical stimulation, repeating every 10 seconds for the duration of the session. No stimulation was delivered when the subjects were restricted to the dark chamber. 24 hours after the final conditioning session, mice were again allowed to explore the entire apparatus without stimulation for 15 minutes. D, During the baseline test, mice show a marked preference for the dark chamber. This preference is noticeably reduced after conditioning. The heat maps depict the average sessions for all mice tested. E, After conditioning the mice changed their preference for the dark chamber (N = 13; 11 with bilateral, and 2 with unilateral fiber optic implants, p<0.0001) vs the lighted one and, on average, showed a preference for the lighted chamber (p=0.0092). GFP control mice that underwent the same conditioning treatment maintained their bias for the dark chamber (N=10, p<0.0001 before and after). Therefore, the optogenetic conditioning had a significant effect on the ChR2 expressing mice (pre vs post p<0.0001) but not in the GFP expressing mice (pre vs post p>0.9999). (Two way ANOVA followed by Bonferroni post-hoc test. Data are mean ± SD).

Fig. 5.. Manipulating the activity of cerebellar axons in the VTA alters social preference

A, ArchT was expressed in the DCN and fiber-optics were bilaterally implanted targeting the VTA to allow optogenetic inhibition of cerebellar axons. B+C, Experimental paradigm. Mice were tested using a three chamber social task. Mice were allowed to approach a juvenile confined to one side chamber or an object placed on the opposite side chamber. On the first trial day, the mice explored the chambers at will. On the second day, a continuous light was delivered to inactivate the cerebellar axons in the VTA whenever the mouse visited the mouse chamber and was terminated immediately if the mouse exited the mouse chamber. On the third trial day, the mice were allowed to explore the chamber again while receiving continuous light independently of their location in the apparatus and for the entire 10 min trial. D+E, Position heat maps for a single mouse (D) and average for all mice (E) during social interaction, in the absence (top row), and in the presence of optogenetic inhibition of cerebellar axons in the VTA in the mouse chamber (middle row) or in the entire field (bottom row). F, Optogenetic inhibition of cerebellar axons in the VTA while the animal explored the mouse chamber made the mouse chamber less attractive compared to day1 (day1: p<0.0001, day2: 0.5564, N=11). Optogenetic inhibition delivered throughout the three chambers similarly decreased the preference for the social compartment (day3: p>0.9999, N = 23). (Regular and RM Two-Way ANOVA followed by post hoc Bonferroni, data are mean ± SD). G. Inhibition of cerebellar axons in the VTA while the animal explored the mouse chamber slightly increased the number of entries in the object chamber (N=11, p = 0.0193), however the number of entries in both chambers were not significantly affected by continuous light inhibition throughout the apparatus (N=23, p=0.0528). (Two-Way ANOVA followed by post hoc Bonferroni, data are mean ± SD). H, Inhibition of cerebellar fibers in the VTA as the mice performed the three chamber social task did not affect grooming time (N=23, p = 0.1475). (Two-Way ANOVA followed by post hoc Bonferroni, data are mean ± SD).

Fig. 6.. Three-chamber social test: Optogenetic stimulation in the object compartment.

(A) ChR2 was expressed in the DCN and fiber optics were bilaterally implanted targeting the VTA to allow optogenetic activation of cerebellar axons. (B) Stimulation paradigm. A train of 1-ms optical light pulses (20 Hz for 3 s) was delivered to activate the cerebellar axons in the VTA whenever the mouse entered the object chamber. This optical train was repeated every 10 s as long as the mouse remained in the object chamber, and was terminated immediately if the mouse exited the object chamber. (C) Experimental paradigm. Mice were tested using a three-chamber social task. Mice were allowed to approach a juvenile confined to one side chamber or an object placed on the opposite side chamber. On the first trial day, the mice explored the chambers at will. On the second day, mice received optogenetic stimulation in the object chamber as described in (B). (D and E) Position heat maps for a single mouse (D) and average for all mice (E) during social interaction, in the absence (left) and in the presence of optogenetic activation of cerebellar axons in the VTA (right) in the object chamber. (F) On day 1, during baseline testing, all groups preferred spending time in the mouse chamber rather than in the object chamber (ChR2, N = 15, GFP N = 12). On day 2, optogenetic activation of cerebellar axons in the VTA while the animal explored the object chamber made the object chamber slightly more attractive than the social chamber housing the juvenile mouse (N = 15). The same treatment did not produce any change in preference in sham GFP mice (N = 12). Data are means ± SD of time spent in the three chambers (two-way ANOVA followed by Bonferroni post hoc test). (G) Activation of cerebellar axons in the VTA while the animal explored the mouse chamber did not affect the number of entries in the social or in the object chamber (N = 15). Similarly, sham GFP mice were not affected by the laser stimulation (N = 12). Data are means ± SD (two-way ANOVA followed by Bonferroni post hoc test). (H) Activation of cerebellar fibers in the VTA as the mice performed the three-chamber social task slightly decreased grooming time relative to baseline, although not significantly (N = 15). Grooming was not affected by laser stimulation in the GFP group (N = 12). Data are means ± SD (two-way ANOVA followed by Bonferroni post hoc test). (I) Mice were allowed to freely interact with a juvenile mouse in an open field and received trains of stimulation every 10 s for 10 min. (J to M) Activation of cerebellar fibers in the VTA while the mice were free to interact in an open field did not significantly affect nose-nose (K) or nose-body interactions (L), following behavior (M), or total investigations (J) in ChR2-expressing mice (N = 7) relative to GFP-expressing mice (N = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7.. Calcium activity in cerebellar axons in the VTA increases as the mice explore the social chamber.

(A) Fiber photometry was used to monitor activity of cerebellar axons in the VTA. GCaMP6 was expressed in the DCN and an imaging fiber-optic was implanted in the VTA. Mice were tested on the same three-chamber social task described in Fig. 5, and changes in GCaMP6 fluorescence in the axons were monitored. (B and C) Mice showed greater GCaMP6 fluorescence in cerebellar axons while they explored the social chamber. (B) Single-trial example. (C) Group average of the photometry session (N = 8). Top row: Total GCaMP fluorescence with respect to position in the chamber. Bottom row: Time spent by the mouse with respect to position in the chamber during the test. (D) Average GCaMP fluorescence per position pixel in the Average GCaMP fluorescence per position pixel in the social chamber correlated with the percent of time spent in the social chamber for each mouse (N = 8, R = 0.904). (E) Average GCaMP fluorescence per position was greater in the social chamber than in the object chamber. Fluorescence values for each chamber in the fluorescence heat maps in (C) were averaged and normalized to the fluorescence in the object chamber. There was significantly greater fluorescence in the social and central chambers between GCaMP-expressing mice (N = 8) and GFP-expressing mice (N = 7). Within the GCaMP group, there was significantly greater fluorescence between the social and central chambers relative to the object chamber (two-way ANOVA followed by Bonferroni post hoc test). *P < 0.05.