Abstract
Evidence of direct reciprocal connections between the cerebellum and basal ganglia has challenged the long-held notion that these structures function independently. While anatomical studies have suggested the presence of cerebellar projections to the substantia nigra pars compacta (SNc), the nature and function of these connections (Cb–SNc) is unknown. Here we show, in mice, that Cb–SNc projections form monosynaptic glutamatergic synapses with dopaminergic and non-dopaminergic neurons in the SNc. Optogenetic activation of Cb–SNc axons in the SNc is associated with increased SNc activity, elevated striatal dopamine levels and increased locomotion. During behavior, Cb–SNc projections are bilaterally activated before ambulation and unilateral lever manipulation. Cb–SNc projections show prominent activation for water reward and higher activation for sweet water, suggesting that the pathway also encodes reward value. Thus, the cerebellum directly, rapidly and effectively modulates basal ganglia dopamine levels and conveys information related to movement initiation, vigor and reward processing.
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Data availability
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact K.K.
Code availability
All data reported and the analysis codes used in this study are available upon request.
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Acknowledgements
The work was funded, in part, by R01MH115604 and R01DA044761 (K.K.). F.N. was supported in part by R01MH060605. We thank the members of the Khodakhah laboratory and S. Nicola for helpful discussions and feedback. dLight used for dopamine measurements was a kind gift from L. Tian. Rabies and helper viruses for retrograde tracing were kind gifts from A. Hantman and K. Ritola. The Zeiss LSM 880 Airyscan confocal microscope of the Neural Cell Engineering and Imaging Core was purchased by funds provided by the NIH Shared Instrument Grant S10OD025295.
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S.W. and R.B. performed in vivo electrophysiology experiments and post hoc histology. S.W. performed in vitro electrophysiological recordings and post hoc histology. M.O. performed anatomical tracing experiments. Image analyses were performed by M.O. and L.K. J.V. performed dopamine measurements and fiber photometry experiments in head-restrained animals on the wheel, with post hoc histology performed by M.O. and J.V. The Pavlovian task and its fiber photometry were performed by J.Y., and post hoc histology was performed by L.K. F.N. supervised and contributed to the analysis of behavioral experiments. K.K. supervised all aspects of the research and secured funding.
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Extended data
Extended Data Fig. 1 Post-hoc histology of in vivo electrophysiology experiments, related to Fig. 1.
(A) AAV-ChR2-EYPF was injected into the DCN and an optrode was placed in the SNc for in vivo recording. (B) (Top, left) Schematic of SNc region zoom in. (Top, right) Expression of ChR2 in cerebellar fibers in SNc was visualized with YFP (green). TH staining was performed to delimit the structure and to label for dopaminergic neurons (blue). Scale bar: 250 µm. (Bottom) Different examples showing cerebellar axons in SNc. Scale bars: 100 µm. (C) (Top) Example of the lesion in the tissue caused by the optrode in the SNc. (Bottom) To track the position of the optrode, it was labeled with DiI (red). Scale bars: 250 µm.
Extended Data Fig. 2 Increasing the strength of optogenetic activation of Cb-SNc axons in vivo increases the response of SNc neurons, related to Fig. 1.
(A) The response of SNc neurons increased with increasing laser intensities. Example response of a neuron to increasing intensities of light stimulation. (B) Summary of cells recorded from SNc under stimulation with various light intensities.
Extended Data Fig. 3 Post-hoc histology of dopamine measurements experiments, related to Fig. 1.
(A) AAV-ChR2-EYFP was injected in the DCN and AAV-dlight1.1 in the striatum. Optic fibers were implanted in the SNc to stimulate Cb-SNc axons and in the striatum to measure dopamine fluctuations. (B) (Left) Schematic of SNc region zoom in. (Right) Optic fiber location in SNc. Scale bar: 250 µm. (C) (Left) Schematic of striatum region zoom in. (Right) Optic fiber location in striatum. Scale bar: 1 mm.
Extended Data Fig. 4 Raw data and onset latency of striatal dopamine evoked by Cb-SNc activation, related to Fig. 1j,k.
(A) Mice were injected with ChR2 in the DCN, and the dopamine sensor dLight1.1 in the striatum. Optic fibers were implanted in SNc to deliver optogenetic pulses and in the dorsolateral striatum to monitor dopamine fluctuations. (B) Raw data traces of dopamine dLight1.1 signals recorded in the dorsolateral striatum. Both spontaneous and optogenetically-evoked (single 1 ms pulses, top or 20 Hz trains, bottom) events are present. (C) (Left) Dopamine signal Z-score aligned to the time of optogenetic stimulation (1 ms pulse at 1 mW, mean ± SEM, n = 7 fibers, N = 4 mice). (Right) Expansion of the first data point after optogenetic stimulation (dotted box). Open circles are the mean Z-score of all the trials (128) in each mouse. One-sided paired t-test of the mean data points obtained just before and just after optogenetic stimulation.
Extended Data Fig. 5 Anatomical evidence for monosynaptic projections from the DCN to the SNc, related to Fig. 3.
(A) Experimental design. RCE:loxp mice were injected bilaterally with AAV1.Cre to label DCN-recipient cells in the SNc. (B) Representative image of AAV1-Cre transfected neurons in the three DCN. DN: dentate nucleus, IntN: interposed nucleus, FN: fastigial nucleus, VN: vestibular nucleus. Scale bar: 250 µm. (C) Average heatmap of the DCN AAV1-Cre transfection efficacy (N = 3). Scale bar: 250 µm. (D) Representative image of the SNc. TH positive neurons are labeled in red, SNc neurons receiving inputs from the DCN are labeled with GFP in green, nuclei are labeled with DAPI in blue. Both TH-positive (arrow) and TH-negative (arrowhead) SNc neurons were targeted by the DCN. Scale bars: 50 and 25 µm. (E) Locations of all DCN-targeted neurons along the SNc. N = 3 mice. Scale bar: 200 µm. (F) Number of DCN-targeted neurons in the SNc along the antero-posterior axis. Neuronal counts from both hemispheres in each section of the SNc were combined. Mean ± SEM, N = 3.
Extended Data Fig. 6 Post-hoc histology of dopamine measurements in DLS, related to Fig. 5.
(A) AAV-ChR2-EYFP was injected in the DCN and AAV-dlight1.1 in the striatum. Optic fibers were implanted in the SNc to stimulate Cb-SNc axons and in the striatum to measure dopamine fluctuations. (B) (Left) Schematic of SNc region zoom in. (Right) Optic fiber location in SNc.ChR2 is shown in green (YFP), TH in red and nuclei is blue (DAPI). Scale bar: 200 µm. (C) (Left) Schematic of striatum region zoom in. (Right) Optic fiber location in dorsolateral striatum. Scale bar: 200 µm.
Extended Data Fig. 7 Post-hoc histology of simultaneous fiber photometry recordings of Cb-SNc axons and SNc neurons in mice on the wheel, related to Fig. 6a–c.
(A) AAV-GCaMP7 was injected in the DCN and AAV-jRGECO1 was injected in the SNc. Optic fiber was implanted in the SNc for dual recordings. (B) Schematic of SNc region. (C) (Left) Expression of jRGECO and fiber location in SNc. Scale bar: 1 mm. (Right) A zoom in of the image shows jRGECO neurons in red. TH was used to delimit SNc area (blue). Optic fiber was located above the SNc. Scale bar: 250 µm.
Extended Data Fig. 8 Post-hoc histology of single fiber photometry recordings in mice performing the lever-manipulation and Pavlovian tasks, related to Fig. 7d,e and 7a–c.
(A) AAV-GCaMP7 was injected in the DCN. Optic fibers were implanted in the SNc to measure the Cb-SNc activity. (B) (Left) Schematic of SNc region. (Right) Optic fiber location in SNc. Scale bar: 1 mm.
Extended Data Fig. 9 Reward-related activity of cerebellar axons in the SNc is bilateral, related to Fig. 7a,b.
(A) Schematic of bilateral recording of Cb-SNc axons during regular reward consumption. (B) GCaMP signals from Cb-SNc axons recorded simultaneously from the right (top) and the left (bottom) SNc during regular reward trials. Mean ± SEM.
Extended Data Fig. 10 Post-hoc histology of dual fiber photometry recordings in mice performing a Pavlovian task, related to Fig. 7c–f.
(A) AAV-GCaMP7 was injected in the DCN and AAV-FLEX-jRGECO1 in the SNc of DAT-Cre mice. An optic fiber was implanted in the SNc to measure Cb-SNc axons and SNc-DA neurons activity while an optic fiber in the dorsolateral striatum (DLS) recorded SNc-DA neurons axons. (B) Schematic of SNc region (zoom in). Expression of jRGECO and fiber location in SNc. jRGECO neurons (in red) colocalized with TH (blue) and Cb-SNc fibers expressing GCaMP (green). Optic fiber was located above the SNc. Scale bar: 250 µm. (C) Schematic of striatum (zoom in). Optic fiber location in DLS. Scale bar: 1 mm.
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Washburn, S., Oñate, M., Yoshida, J. et al. The cerebellum directly modulates the substantia nigra dopaminergic activity. Nat Neurosci 27, 497–513 (2024). https://doi.org/10.1038/s41593-023-01560-9
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DOI: https://doi.org/10.1038/s41593-023-01560-9
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