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A cortico-cerebellar loop for motor planning

Naturevolume 563pages113116 (2018) | Download Citation

Abstract

Persistent and ramping neural activity in the frontal cortex anticipates specific movements1,2,3,4,5,6. Preparatory activity is distributed across several brain regions7,8, but it is unclear which brain areas are involved and how this activity is mediated by multi-regional interactions. The cerebellum is thought to be primarily involved in the short-timescale control of movement9,10,11,12; however, roles for this structure in cognitive processes have also been proposed13,14,15,16. In humans, cerebellar damage can cause defects in planning and working memory13. Here we show that persistent representation of information in the frontal cortex during motor planning is dependent on the cerebellum. Mice performed a sensory discrimination task in which they used short-term memory to plan a future directional movement. A transient perturbation in the medial deep cerebellar nucleus (fastigial nucleus) disrupted subsequent correct responses without hampering movement execution. Preparatory activity was observed in both the frontal cortex and the cerebellar nuclei, seconds before the onset of movement. The silencing of frontal cortex activity abolished preparatory activity in the cerebellar nuclei, and fastigial activity was necessary to maintain cortical preparatory activity. Fastigial output selectively targeted the behaviourally relevant part of the frontal cortex through the thalamus, thus closing a cortico-cerebellar loop. Our results support the view that persistent neural dynamics during motor planning is maintained by neural circuits that span multiple brain regions17, and that cerebellar computations extend beyond online motor control13,14,15,18.

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Raw and processed data are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank R. Sillitoe for L7-cre mice; J. Medina, H. Inagaki, Z. Guo and M. Ahrens for comments on the manuscript; S. Druckmann for discussion; T. Pluntke and M. Inagaki for animal training; and J. White and H. Hasanbegovic for dystonia scoring. This work was funded by the Robert and Janice McNair Foundation (N.L.), the Whitehall Foundation (N.L.), the Alfred P. Sloan Foundation (N.L.), the Searle Scholars Program (N.L.), the National Institutes of Health NS104781 (N.L.), the Simons Collaboration on the Global Brain (K.S. and N.L.), the Dutch Organization for Medical Sciences (C.I.D.Z.), Life Sciences (Z.G. and C.I.D.Z.), an Erasmus MC fellowship (Z.G.), the ERC-advanced and ERC-PoC (C.I.D.Z.) and the Howard Hughes Medical Institute (K.S., M.E. and N.L.).

Reviewer information

Nature thanks T. Ebner, M. Joshua and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Department of Neuroscience, Erasmus MC, Rotterdam, The Netherlands

    • Zhenyu Gao
    •  & Chris I. De Zeeuw
  2. Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA

    • Courtney Davis
    • , Alyse M. Thomas
    • , Amada M. Abrego
    •  & Nuo Li
  3. Janelia Research Campus, Ashburn, VA, USA

    • Michael N. Economo
    • , Karel Svoboda
    •  & Nuo Li
  4. Netherlands Institute for Neuroscience, Amsterdam, The Netherlands

    • Chris I. De Zeeuw

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Contributions

N.L., K.S., C.I.D.Z. and Z.G. conceived the project. Z.G., N.L. and C.I.D.Z. performed pilot experiments. N.L., Z.G., C.D., A.M.T. and A.M.A. performed behavioural and recording experiments. Z.G. performed anatomy experiments. M.N.E. contributed brain alignment software. N.L., Z.G., K.S. and C.I.D.Z. analysed the data. N.L., Z.G., C.I.D.Z. and K.S. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Nuo Li.

Extended data figures and tables

  1. Extended Data Fig. 1 Histology and behavioural data.

    a, Coronal sections showing CN lesion sites for all mice. Fastigial lesion, n = 4; dentate lesion, n = 4. b, Execution of the licking movement is not affected by fastigial lesions. Individual licks are shown as dots (blue, lick right; red, lick left). Licks are aligned to the onset of the ‘go’ cue (vertical line). c, Average lick rate within a lick bout and the duration of lick bouts are not affected by fastigial lesion. A lick bout is defined as a sequence of licks with an inter-lick interval of less than 500 ms. Data from the fastigial-lesion mice only (n = 4). Data are grouped by licking direction relative to the lesioned hemisphere: contralesional licking (blue) or ipsilesional licking (red). d, Coronal sections showing ChR2–eYFP expression in the fastigial nucleus and the dentate nucleus. Nine mice were used for various fastigial ChR2 photoactivation experiments, and ten mice were used for dentate ChR2 photoactivation experiments. In one of the fastigial photoactivation mice, the injection of ChR2 virus missed the CN (not shown) and its data were excluded. Two of the dentate photoactivation mice were subsequently used for lesion experiments (not shown). See Supplementary Table 1 for the list of mice and manipulated hemispheres in individual experiments. e, CN photoactivation during specific behavioural periods. Top, the experiment timeline. Photostimulation was for the entire task period (1.3 s). Bottom, performance in the behavioural trials. Left, fastigial photoactivation (n = 6); right, dentate photoactivation (n = 8). For fastigial photoactivation, average behavioural performance for control and photoactivation trials (S, sample period photoactivation; D, delay period; R, response period) is also shown. Grey lines represent individual mice. Chance is 50%. For behavioural performance grouped by trial type, lick-left and lick-right trials are grouped by instructed licking direction relative to the manipulated hemisphere. Blue, contralateral; red, ipsilateral. Both hemispheres were tested. See Supplementary Table 1 for the list of mice and manipulated hemispheres. Thick lines represent the mean; thin lines represent individual mice (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, one-sided test, bootstrap (Methods). f, Proportion of early-lick and no-lick trials with and without CN photoactivation during the delay period. Control trials are shown in grey and photoactivation trials in cyan. Lines represent individual mice. FN, fastigial photoactivation (n = 6); DN, dentate photoactivation (n = 8).

  2. Extended Data Fig. 2 Characterization of CN photoinhibition and behavioural data.

    a, Strategy for silencing the CN. ChR2 is expressed in cerebellar Purkinje cells, and the photostimulation of Purkinje cells inhibits CN neurons (photoinhibition). b, Top left, an image of the cerebellar cortex showing the expression of ChR2–eYFP in Purkinje cells in a L7-cre × Ai32 mouse. Top right, an example silicon-probe recording in a L7-cre × Ai32 mouse. The coronal section shows ChR2–eYFP expression in the cerebellar cortex and DiI-labelled electrode tracks. Bottom, electrode tracks within the fastigial nucleus (dashed contour). Photostimulation of Purkinje cells was carried out by directing a blue laser (0.5–4.5 mW average power, beam diameter 400 µm at 4σ) to the brain surface through a cranial window. c, Example voltage traces from a recording of the fastigial nucleus during photoinhibition. Multi-unit activity in the fastigial nucleus was silenced. Multiple traces show repeats of photostimulation. d, Photoinhibition reduced activity specifically in the CN. Average spike rates before (500 ms) versus during photoinhibition (1.2 s) are shown, for neurons from the fastigial nucleus (left, n = 26) and from the surrounding cerebellar cortex (right, n = 34). Photostimulation of the cerebellar cortex (lambda posterior 3 mm, lateral 2 mm) silenced fastigial activity whereas photostimulation of the inferior colliculus (posterior 1 mm, lateral 2 mm) did not. Solid dots indicate neurons with a significant change in spike rate (P < 0.01, two-tailed t-test). e, Dose-dependent effect of CN photoinhibition on posture and movement. Left, video frames showing an unrestrained mouse undergoing CN photoinhibition. During low-intensity photoinhibition (1.5 mW, the intensity typically used in the delayed response task), no consistent movement was detected. High-intensity photoinhibition (20 mW) of both the fastigial nucleus and the dentate nucleus produced dystonia-like movements and posture changes, such as loss of balance and extensions of the contralateral limbs (arrows). Right, dystonia-like movements observed upon photoinhibition were given scores on a scale of 0–4 (Methods): 0, no motor abnormalities; 1, slightly slowed or abnormal motor behaviour, no dystonia; 2, mild impairment, mild and transient dystonic postures, weak tremor; 3, moderate impairment, dystonic postures, cannot balance the body, major tremor; 4, severe impairment, sustained dystonic postures and limited movements. The scoring was performed blind to the experimental conditions. Data are mean ± s.e.m. across trials. n = 2 mice, 10 trials per condition. f, Dose-dependent effect of CN photoinhibition on jaw movement and eyeblink. Top, video frames showing a head-fixed mouse undergoing CN photoinhibition. Bottom left, eyeblink responses during CN photoinhibition. No consistent eyeblink was evoked. Bottom right, jaw movements during CN photoinhibition. Only high-intensity photoinhibition (20 mW) of the dentate nucleus produced jaw movements. Data are mean ± s.e.m. across trials. n = 4 mice, 10 trials per condition for eyeblink, 20 trials per condition for jaw movement. g, Behavioural performance in the delayed response task with and without fastigial photoinhibition for the entire task periods (n = 3). Left, average behavioural performance for control and photoinhibition trials (S, sample period photoinhibition; D, delay period; R, response period). Grey lines show individual mice. Chance is 50%. Right, behavioural performance for each trial type. Lick-left and lick-right trials are grouped by instructed licking direction relative to the manipulated hemisphere. Blue, contralateral; red, ipsilateral. Both hemispheres were tested. See Supplementary Table 1 for the list of mice and manipulated hemispheres. Thick lines represent the mean; thin lines represent individual mice. *P < 0.05, **P < 0.01, ***P < 0.001, one-sided test, bootstrap (Methods). h, Behavioural performance in trials with photoinhibition during the early sample, late sample, early delay, and late delay periods (n = 7 mice). *P = 0.04, ***P = 0.0002, one-sided test, bootstrap. i, Proportion of early-lick and no-lick trials with fastigial photoinhibition during the delay period. Control trials (grey) and photoinhibition trials (cyan). Lines, individual mice (n = 7).

  3. Extended Data Fig. 3 Silicon-probe recordings in the CN.

    a, DiI-labelled recording tracks in coronal sections stained by DAPI. Borders of the CN are visible in DAPI staining. Electrode location was estimated based on DiI labelling and manipulator depth. The lamination of activity patterns across the electrodes corresponded well to the anatomical structures: that is, high activity levels in the CN and the cerebellar cortex, low activity levels in the white matter. Single-unit locations were estimated on the basis of electrode location. Top, a coronal section showing a recording in the fastigial nucleus. Bottom, the fastigial nucleus (yellow dashed line), estimated electrode location, and the locations of single units (units 1–6) from an example recording. b, Left, voltage trace from the recording site of unit 1 during a single behavioural trial. Dashed lines indicate behavioural periods. Right, waveforms of unit 1 after spike sorting. c, Peristimulus time histogram of all the units from the example fastigial recording. The dashed box indicates that units 1–5 were within the fastigial nucleus, and unit 6 was outside of the fastigial nucleus. Blue, lick-right trials; red, lick-left trials. d, Same as a and c, but for an example recording in the interposed nucleus. e, Same as a and c, but for an example recording in the dentate nucleus.

  4. Extended Data Fig. 4 ALM and CN activity is related to motor planning and movement.

    a, Criteria used to identify neuronal activity modulated by licking. For each lick, spike counts were calculated in two adjacent 50-ms time windows, starting 20 ms from the detection time of the lick. Across all licks and all trials, neurons with significant difference in spike count between the two time windows were deemed to be modulated by licking (P < 0.01, two-tailed t-test). Licking modulation is calculated for each neuron: mean difference in spike rate between the two time windows. b, Example neurons are significantly modulated by licking. Peristimulus time histogram from the correct lick-right and lick-left trials during the response period. Spike times are aligned to the first lick. Trials are grouped by licking direction relative to the recorded hemisphere: contralateral (blue) or ipsilateral (red). Averaging window, 10 ms. c, Fraction of neurons that are significantly modulated by licking (P < 0.01, two-tailed t-test) from each brain region. FN, fastigial nucleus; IP, interposed nucleus; DN, dentate nucleus. Error bars indicate s.e.m. across mice, bootstrap (Methods); dots represent individual mice (ALM, n = 10; FN, n = 15; IP, n = 9; DN, n = 11). d, There is no correlation between whether a neuron was modulated by licking and whether it exhibited preparatory activity. Top, ALM; bottom, the cerebellar nuclei. Selectivity during the delay period is the difference in spike rate between the lick-right and lick-left trials. Solid dots indicate neurons that are significantly modulated by licking (P < 0.01, two-tailed t-test). e, Preparatory activity in the CN. Left, Peristimulus time histograms for correct and error trials are shown for two example CN neurons. Trial types are based on sensory instruction (blue, lick right; red, lick left). The same trial-type preference in correct and error trials indicates selectivity for object location. Opposite trial-type preference indicates selectivity for upcoming movement directions. A negative ratio of trial-type selectivity between error and correct trials means a neuron switches trial-type preference to predict upcoming movement directions on error trials. Right, selectivity ratios for CN neurons. Neurons with significant delay-period selectivity and tested for >3 error trials are shown (n = 73). f, Population selectivity for each cerebellar nucleus (mean ± s.e.m. across neurons, bootstrap; fastigial, n = 87; interposed, n = 50; dentate, n = 60). Selectivity is the difference in spike rate between the preferred and the non-preferred trial type (Methods). Dashed lines separate sample, delay and response periods. g, Proportion of contra-preferring versus ipsi-preferring neurons, based on spike counts during the first 500 ms (left) and the last 500 ms (right) of the delay period. Error bars indicate s.e.m. across mice. Dots represent individual mice (ALM, n = 10; FN, n = 15; IP, n = 9; DN, n = 11). ***P = 0.0001, one-sided test, bootstrap.

  5. Extended Data Fig. 5 ALM drives CN preparatory activity and vice versa.

    a, Top, schematic showing CN recording during bilateral ALM photoinhibition. Laser power, 1.5 mW per location (Methods). Bottom, Peristimulus time histograms of four example CN neurons with and without ALM photoinhibition. The cyan bar indicates the photoinhibition period. Blue, lick-right trials; red, lick-left trials. b, Left, average fastigial selectivity in control and photoinhibition trials (mean ± s.e.m. across neurons, bootstrap). Only selective neurons tested for >3 trials in all conditions are included (n = 54 neurons, 8 mice). Orange dashed line denotes the mean from control trials. Right, relationship between delay-period selectivity of individual fastigial neurons and changes in firing rate due to ALM photoinhibition. Filled circles indicate neurons that are significantly modulated by ALM photoinhibition (P < 0.01, two-tailed t-test). c, Same as b, but for the interposed nucleus (n = 44 neurons, 5 mice). d, Same as b, but for the dentate nucleus (n = 59 neurons, 6 mice). e, Same as a, but for ALM recording during contralateral CN photoinhibition. Laser power, 1.5–4.5 mW.

  6. Extended Data Fig. 6 CN preparatory activity is driven by both hemispheres of the ALM.

    a, CN recording during contralateral or ipsilateral ALM photoinhibition. b, Performance in the delayed response task during unilateral and bilateral ALM photoinhibition. c, CN population selectivity from control and photoinhibition trials (mean ± s.e.m. across neurons, bootstrap). Only selective neurons tested for >3 trials in all conditions are included (n = 157). The orange dashed line indicates the mean from control trials. The cyan bar denotes the photoinhibition period. CN selectivity was reduced by photoinhibiting either side of the ALM. d, Peristimulus time histograms of example CN neurons during ipsilateral or contralateral ALM photoinhibition. The effect was heterogeneous across individual neurons. Some neurons were affected by ipsilateral ALM photoinhibition (row 1–3), other neurons were affected by contralateral ALM photoinhibition (row 4), others were affected by photoinhibition of either side (row 5, 6). e, Peristimulus time histograms of example CN neurons during bilateral ALM or M1 photoinhibition. ALM: bregma anterior 2–3 mm, lateral 1–2 mm; M1 anterior 0–1 mm, lateral 1–2 mm.

  7. Extended Data Fig. 7 Alignment of anatomical data and comparing fastigial and dentate projections to ALM-projecting thalamus.

    a, Top, an example coronal section before and after alignment to the corresponding section of the anatomical template in the Allen Mouse Common Coordinate Framework (CCF). Fluorescence is anterograde labelling (BDA) from an ALM injection, labelling the ALM-projecting thalamus (Methods). Yellow dots indicate control points for b-spline transformation. The dashed line denotes a region of interest containing the thalamus ipsilateral to the ALM injection. Bottom, overlay of the aligned image (orange) and the anatomical template (green). ALM axons in the thalamus obey structural boundaries in the anatomical template. Critically, the alignment procedure did not use any structures inside the thalamus as landmarks. b, Coronal sections showing an example fastigial injection case with anterograde labelling in the thalamus. The sections are aligned to and superimposed on the anatomical template. The yellow outlines show labelled areas after thresholding (Methods). c, Coronal sections showing three different fastigial injection cases. Different injections show similar patterns of anterograde labelling in the thalamus. d, Left, outlines of the labelled areas from all fastigial injection cases (n = 6). Borders of the thalamic nuclei (VM and VAL) are based on annotations in the Allen Reference Brain. Right, outlines of the labelled areas from all fastigial (n = 6), dentate (n = 4) and ALM (n = 8) injection cases. There was a greater overlap between fastigial and ALM labelling. e, Total areas labelled (number of pixels) in the thalamus by fastigial, dentate and ALM injections. Dots indicate individual coronal sections; lines represent individual injections. f, Overlaps between fastigial or dentate projections and the ALM-projecting thalamus. The co-labelled area is normalized to the total thalamic area labelled by fastigial or dentate projections (area fraction overlap). Dots indicate individual coronal sections; lines represent individual injections.

  8. Extended Data Fig. 8 The ALM-projecting thalamus receives converging inputs from the fastigial nucleus, basal ganglia SNr and the superior colliculus.

    a, Example triple injections showing co-labelling of fastigial and dentate projections (left), fastigial and SNr projections (middle), and fastigial and superior colliculus projections (right) in the thalamus. Injections were repeated several times in different mice with similar results (ALM–fastigial–dentate, n = 4; ALM–fastigial–SNr, n = 3; ALM–fastigial–superior colliculus, n = 2). b, Overlaps between ALM-projecting thalamus and projections of different areas (Methods). Fastigial, SNr and superior colliculus labelling show a comparable amount of overlap with the ALM-projecting thalamus. Dentate labelling shows less overlap. The thalamic area co-labelled by ALM injection and a projection (fastigial, dentate, SNr or superior colliculus) is normalized to the total thalamic area labelled by that projection (area fraction). Dots indicate individual coronal sections; lines represent individual injections. c, A confocal image showing vGlut2 staining in the ALM-projecting thalamus. Anterograde tracer injections in the ALM (blue), the fastigial nucleus (green) and the SNr (red). Fastigial axons form glutamatergic synapses (vGlut2 positive) within the ALM-projecting thalamus. vGlut2 staining was performed in one ALM–fastigial–SNr injection case.

  9. Extended Data Fig. 9 CN ChR2 photoactivation drives rapid change in ALM activity.

    a, The change in firing rate of ALM neurons during fastigial or dentate ChR2 photoactivation. The change in firing rate is the difference in spike rate between control and photoactivation trials. For neurons that were suppressed by CN photoactivation, the firing rate differences are multiplied by −1 so that the firing rate changes are always positive for latency quantifications. Data are mean ± s.e.m. across neurons. Only neurons that were significantly modulated by CN photoactivation are included (P < 0.01, two-tailed t-test; fastigial photoactivation, n = 227; dentate photoactivation, n = 163). Top, Time course for the change in ALM firing rate. The cyan bar denotes the CN photoactivation period (500 ms). Bottom, The onset of the change in firing rate of ALM neurons (arrows). The change of firing rate is quantified in 10-ms time bins in 1-ms steps. The onset time is the first time bin in which the change in population firing rate significantly deviated from zero (P < 0.01, two-tailed t-test). We repeated the onset time estimation 10,000 times. In each round, we resampled with replacement from the neurons in the dataset and re-estimated the onset time. The arrows indicate the mean estimated onset time. b, Relationship between selectivity of individual ALM neurons and changes in firing rate due to CN photoactivation. Filled circles denote ALM neurons that were significantly modulated by CN photoactivation (P < 0.01, two-tailed t-test). The change in firing rate is the difference in the spike rate between control and photoactivation trials during photostimulation (top) or the last 500 ms of the delay period (bottom). The selectivity is the difference in spike rate between lick-right and lick-left trials during the delay period. c, Selectivity of the ALM population from control and photoactivation trials (mean ± s.e.m. across neurons, bootstrap; selective neurons tested for >3 trials in all conditions). The orange dashed line indicates the mean from control trials. Top, fastigial photoactivation (n = 328); bottom, dentate photoactivation (n = 377). The cyan bar denotes the photoactivation period.

  10. Extended Data Fig. 10 Fastigial photoactivation abolishes the relationship between ALM activity and upcoming movements.

    a, ALM recording during bilateral ALM photoinhibition (top) or contralateral fastigial photoactivation (bottom). b, Performance in control (grey) and photostimulation (cyan) trials. Lick-left and lick-right trials are pooled. Chance is 50% (dashed line). Lines represent individual mice (ALM photoinhibition, n = 7; fastigial photoactivation, n = 6). ***P < 0.001, one-sided test, bootstrap (Methods). c, Left, schematic of ALM activity projected onto the coding direction (cd). Dashed lines indicate control trials, ALM activity trajectories converge to discrete endpoints at the end of the delay period that predict upcoming movement directions (lick right, blue; lick left, red). Solid black lines indicate bilateral ALM photoinhibition trials; the distance of the perturbed trajectories to the endpoints predicts future movement directions even though the behavioural performance is close to that which would be obtained by chance. Right, experimental data. Probability of the mouse licking right as a function of trajectory distance to the endpoints along the cd (Methods). Bilateral ALM photoinhibition trials only (n = 690). The cd and endpoints were estimated from independent control trials. Data are binned along the cd projection. The s.e.m. is obtained by bootstrapping the trials in each bin. d, Same as c, but for fastigial photoactivation trials (n = 337). ALM activity no longer predicts future movement directions after a fastigial perturbation. e, It is possible that fastigial perturbation activated downstream motor circuits that could maintain the motor plan and generate movements independent of ALM. We examined the necessity of ALM activity in driving directional licking after a fastigial perturbation. In VGAT-ChR2-eYFP mice expressing ChR2 in both Purkinje neurons and cortical GABAergic neurons, we independently manipulated activity in the fastigial nucleus and the ALM (Methods). Left, unilateral ALM photoinhibition during the late delay period. Right, behavioural performance for each trial type in control and photoinhibition trials. Left ALM photoinhibition biased upcoming licking to the left, resulting in lower performance in the lick-right trials. The opposite pattern of bias was induced by right ALM photoinhibition. Thick lines represent the mean; thin lines represent individual mice (n = 4). ***P = 0.0002, one-sided test, bootstrap (Methods). f, Same as e, but for unilateral ALM photoinhibition after fastigial perturbation. Note the same pattern of behavioural bias as e. *P = 0.04, **P = 0.008, one-sided test, bootstrap. g, Left, after fastigial perturbation, ALM activity was bilaterally silenced during movement initiation. Right, fraction of trials in which mice did not lick after the ‘go’ cue. FN, fastigial perturbation only; ALM, bilateral ALM photoinhibition; FN + ALM, fastigial perturbation followed by bilateral ALM photoinhibition. Thick lines represent the mean; thin lines represent individual mice (n = 3). **P = 0.002, ***P = 0.0002, one-sided test, bootstrap.

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