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A cerebello-olivary signal for negative prediction error is sufficient to cause extinction of associative motor learning


The brain generates negative prediction error (NPE) signals to trigger extinction, a type of inhibitory learning that is responsible for suppressing learned behaviors when they are no longer useful. Neurons encoding NPE have been reported in multiple brain regions. Here, we use an optogenetic approach to demonstrate that GABAergic cerebello-olivary neurons can generate a powerful NPE signal, capable of causing extinction of conditioned motor responses on its own.

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Fig. 1: Inhibiting the IO in vivo.
Fig. 2: CNIO stimulation during eye puff stimulus.
Fig. 3: CNIO stimulation after eye puff stimulus.

Data availability

The data that support the findings of this study are available at

Code availability

The codes used to analyze the data in this study are available at


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This study was supported by grants to J.F.M. from the National Institutes of Health (grant nos. R01 MH093727, RF1 MH114269 and NIH R01NS112917), and a grant to O.A.K. (grant no. F31 NS103427). We thank S. Heiney for help with surgical approach and J. Siegel for help with analysis.

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Authors and Affiliations



O.A.K. and J.F.M. designed the experiments. J.F.M. supervised the project. O.A.K. performed virus injection and optical fiber implant surgeries, and conducted all behavior/optogenetics experiments. S.O. performed all electrophysiology-related surgery and experiments. O.A.K. analyzed and curated all original data collected for this publication. S.O. curated data included from a previous publication. O.A.K. and J.F.M. wrote the original draft of the paper. J.F.M., O.A.K. and S.O. revised and edited the paper.

Corresponding author

Correspondence to Javier F. Medina.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks John Freeman, Kamran Khodakhah and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Summary of failed experiments using inhibitory opsins in the dorsal accessory olive.

af, Arch expression (green) in and around the IO (outlined in white) in histological sections counterstained with red fluorescent nissl (magenta). Expression of ArchT was very sparse and did not cover the region of the IO involved in eyeblink conditioning (dorsal accessory olive, DAO; white arrows) in Pdx-cre mice crossed with flex-ArchT mice (a), or Pdx-cre mice that received an AAV-ArchT injection in the IO (b). (c-e) Expression of Arch3 was patchy and did not cover the DAO in CRH-cre mice crossed with either heterozygous (c) or homozygous flex-Arch3 mice (e), or in CRH-cre mice that received an AAV-Arch injection in the IO (d). (fi) Although we obtained good expression of Arch in the DAO in wildtype mice injected with AAV1/9-αCaMKII-Arch-GFP (f), the health of 8/11 mice began to deteriorate two weeks after AAV injection, such that the mice could not tolerate eyeblink conditioning sessions and had to be euthanized. Experiments were not possible in the few mice that survived (3/11 mice) because these mice were severely impaired in eyeblink conditioning g, and the performance of even the best mouse (i) was much worse than the performance of control mice (h, control data previously published in21). Abbreviations: CR, conditioned response; CS, conditioned stimulus; DAO, dorsal accessory olive; DM, dorsomedial cell column; FEC, fraction eyelid closure; MAO, medial accessory olive; PO, principal olive; RF, reticular formation.

Extended Data Fig. 2 Olivo-cerebellar circuits relevant to eyeblink conditioning.

Somatosensory information about the eye puff stimulus crosses the midline and is sent to the contralateral inferior olive via the trigeminal nucleus. The inferior olive sends a predominantly contralateral projection to eyeblink-generating Purkinje cells in the cerebellar cortex via the climbing fiber pathway. Purkinje cells and cerebellar nucleus projection (CNRN) neurons control CR generation for the ipsilateral eye. Cerebellar nucleo-olivary neurons (CNIO) send a GABAergic projection to the inferior olive. During the experiments described in this paper, we induced broad ChR2 expression in the cerebellar nuclei and then selectively activated the cerebello-olivary pathway by photostimulating CNIO axon terminals at the level of the inferior olive. Note that CNIO neurons are a distinct population, completely separate from the CNRN neurons, and for this reason, photostimulation-driven backpropagating action potentials in the CNIO axons do not have direct access to the neurons that are responsible for generating the eyeblink CR.

Extended Data Fig. 3. Opsin expression and optical fiber placement in ChR2 and control mice.

a, e, Outline of nuclei around the virus injection site (a) and the fiber implant site (e) traced from a representative mouse (ChR2 1). The cerebellar and inferior olivary nuclei implicated in eyeblink conditioning are highlighted in green (anterior interpositus nucleus, AIP; dorsal accessory olive; DAO) (bd, fh) Coronal sections at the level of the cerebellar nuclei (b-d) and inferior olive fh, from mice in the ChR2 (b, d, f, h) and control (c, g) groups. A unique identifier for each mouse is shown in the bottom left corner of each photomicrograph. Cerebellar and vestibular nuclei are revealed by fluorescent nissl stain (magenta). ChR2-EYFP (green) was visible at the level of the AIP (b-d) and, with longer exposure times, the DAO (fh). The AAV injection did not work in mouse ‘Control 5’. Lesions deliberately made at the end of the experiments to mark the location of the optical fiber tip are also visible just dorsal to the DAO (yellow dashed outline). Abbreviations: AIP, anterior interposed nucleus; cctx, cerebellar cortex; DAO, dorsal accessory olive; DM, dorsomedial cell column of the inferior olive; DC, dorsal cochlear nucleus; DN, dentate nucleus; F, fastigial nucleus; icp, inferior cerebellar peduncle; LVe, lateral vestibular nucleus; MAO, medial accessory olive; MVe, medial vestibular nucleus; P, paracochlear glial substance; PO, principal olive; py, pyramids; RF, reticular formation; VCB, vestibulocerebellar nucleus.

Extended Data Fig. 4 CSpk activity is similar during CNIO stimulation and normal extinction.

a, b, CSpk firing rate (normalized to pre-trial baseline) for individual neurons (heat plots) and for groups of neurons (mean, histograms) in 50-ms bins, aligned to the end of the pause during CNIO stimulation trials in ChR2 mice (a; n = 11 neurons, same as in Fig. 1l) or extinction trials in wildtype mice (b, n = 32 neurons, recorded during a previous experiment5). c, Quantification of rebound size in the 50 ms after the pauses shown in (a, b). Each dot shows the mean, normalized firing rate response in the 50 ms after a pause (rebound window) for a single neuron. Dots are filled if neurons exhibited significant rebound firing (see Online Methods). Of the neurons recorded during CNIO stimulation trials in ChR2 mice, 6/11 (55%) exhibited a significant rebound. Of the neurons recorded during extinction trials in wildtype mice, 15/32 (47%) exhibited a significant rebound. There was no significant difference between rebound firing rates of the neurons recorded in the ChR2 and the wildtype mice (n = 43 neurons, two-sided Wilcoxon rank-sum test: W = 195, p = 0.61; boxplot center: mean, box bounds: ± SEM, whiskers: distribution minimum and maximum).

Extended Data Fig. 5 Extinction during CNIO stimulation displays spontaneous recovery and savings.

a, CR probability (median ± MAD) measured in blocks of 20 trials during the final training session (white background) and repeated CNIO stimulation sessions (gray shaded background) in ChR2 mice (n = 6 mice). Sessions are separated from each other by vertical dotted lines, and the line of best fit to the 5 median values in each session is shown. b, CR probability in the last block of 10 trials in a session was significantly lower than in the first block of 10 trials in the next one for the first 5 sessions with CNIO stimulation for the mice shown in panel a (n = 6 mice; two-tailed paired t-test: t = 3.63, df = 5, p = 0.015, d = 1.55). c, d, Same as (a, b), but for wildtype mice undergoing extinction training (gray shaded background in panel c; n = 5 mice; two-tailed paired t-test: t = 5.53, df = 4, p = 0.005, d = 2.12). e, CR probability (median ± MAD), and f, number of sessions to acquire the task (mean ± SEM), during initial training (filled symbols) and retraining (open symbols) of ChR2 mice before and after CNIO stimulation (n = 6 mice, training: 8 ± 1.06 sessions, retraining: 5 ± 0.86 sessions, one-tailed paired t-test: t = 3.22, df = 5, p = 0.01, d = 1.25). g, h, Same as (e, f), but for initial training and retraining of wildtype mice before and after extinction (h, n = 5 mice, training: 6.5 ± 1.6 sessions, retraining: 3.6 ± 0.68 sessions, one-tailed paired t-test: t = 2.62, df = 4, p = 0.03, d = 0.67). Family-wise alpha values were Bonferroni-Holm corrected for multiple comparisons. In panels b, d, f, h, boxplot center: mean, box bounds: ± SEM, whiskers: distribution minimum and maximum. *p < 0.05.

Extended Data Fig. 6 Extinction during CNIO stimulation is not caused by impaired processing of eye puff stimulus.

a, Averaged reflex eyelid responses to the airpuff (unconditioned response, UR; mean ± SEM). b, UR peak amplitude (n = 7 mice; two-tailed paired t-test: t = 1.41, df = 6, p = 0.21), and c, UR duration in sessions with (blue) and without (black) CNIO stimulation for ChR2 mice (n = 7 mice; two-tailed paired t-test: t = 5.51, df = 6, p = 0.001, d = 1.25). Only trials without eyelid movements preceding the airpuff are included. d, URs (mean ± SEM) from 9 mice, during training sessions in which the intensity of the eye puff was systematically changed to generate long URs (black) or short URs (red). URs during CNIO stimulation sessions in (a) are duplicated for comparison (blue). e, CR probability (open circles) and UR duration (filled circles; mean ± SEM) for the mice in (d), shown during a training session with long URs (black) and the following 5 consecutive training sessions, in which the eye puff intensity was set to generate shorter URs (red). In spite of shorter URs (filled red circles; n = 9 mice, Friedman’s ANOVA: Fr = 12.71, df = 4, p = 0.013, W = 0.35), CR probability remained unchanged (open red circles; n = 9 mice, Friedman’s ANOVA: Fr = 5.07, df = 4, p = 0.28). Family-wise alpha values were Bonferroni-Holm corrected for multiple comparisons. *p < 0.05.

Extended Data Fig. 7 CNIO stimulation before the airpuff does not impair CR performance.

a, Schematic showing relative timing of stimulus presentations and laser pulses in the different phases of the experiment. b, CR probability (median ± MAD) in the last training session (‘TLast’) and sessions during CNIO stimulation (gray shaded area) in ChR2 mice (n = 6 mice; CR probability increased during CNIO stimulation, Friedman’s ANOVA: Fr = 14.86, df = 6, p = 0.02, W = 0.36). c, Averaged eyelid movement traces, and d, CR amplitude during sessions with CNIO stimulation increased significantly (n = 6 mice; Friedman’s ANOVA: Fr = 24.50, df = 6, p = 4.22 × 10−4, W = 0.68). Repeating the analysis in (c-d) but excluding trials without CRs revealed that the increase in CR amplitude during CNIO stimulation shown in (d) is not simply driven by a higher CR probability – CRs were significantly larger (e-f; n = 6 mice; Friedman’s ANOVA: Fr = 20.0, df = 6, p = 0.003, W = 0.56). The time window used to calculate CR amplitude is indicated (c, e; yellow shaded area). g, Averaged eyelid position, and h, eyelid velocity traces for the last training session and subsequent sessions with CNIO stimulation. The gray shaded area highlights the β-startle window, which follows the α-startle and precedes the CR. i, β-startle amplitude was increased (n = 6 mice; Friedman’s ANOVA: Fr = 29.71, df = 6, p = 4.45 × 10−5, W = 0.82), and j, latency was shortened (n = 6 mice; Friedman’s ANOVA: Fr = 25.57, df = 6, p = 2.67 × 10−4, W = 0.71) excluding trials without a β-startle. Family-wise alpha was Bonferroni-Holm adjusted for multiple comparisons. In panels d, f, i, j, boxplot center: mean, box bounds: ± SEM, whiskers: distribution minimum and maximum). **p < 0.01, ***p < 0.001.

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Kim, O.A., Ohmae, S. & Medina, J.F. A cerebello-olivary signal for negative prediction error is sufficient to cause extinction of associative motor learning. Nat Neurosci 23, 1550–1554 (2020).

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