Optogenetic CF co-activation prevents adaptive L2/3 pyramidal cell potentiation in S1 cortex.

(A) Experimental schematic for (D – L). (B and C) ChR2 expression in the inferior olive (IO) and optogenetic CF activation evokes calcium transients in PCs. (B) Calbindin staining of PCs marks the cerebellum (shown here in the same plane as the IO; white arrowhead). Scale bar: 1mm. (C) Two-photon field of view of PC dendrites with calcium responses evoked by 470nm, 50ms LED pulses. Scale bar: 100µm. Right: Distribution of response amplitudes (box: 25th and 75th percentiles; line: median). (D) GCaMP6f expression in S1 cortex. Scale bar: 1mm. Trace scale bar: 0.2 ΔF/F; 0.5s. (E) Sagittal view of ChR2-expressing CF terminals in the contralateral cerebellar cortex that are activated in optogenetic experiments. Scale bar: 100µm. (F) Left: Example of the morphological identification of L2/3 pyramidal cells by apical dendrites (green arrowheads) and INs (white arrowheads) in a two-photon field of view. Scale bar: 100µm. Right: Pyramidal cell at higher magnification. Scale bar: 15µm. (G) Pyramidal cell plasticity after RWS (N=12 mice) and after pairing with CF activation (RWS+CF; N=11 mice). Right: Quantification of evoked responses across neurons. (H) Data in (G) binned across time. Blue shading: RWS/RWS+CF. (I) Quantification across mice, normalized to the pre-plasticity response. (J-L) Analyses for INs in the same mice. Box: 25th and 75th percentiles; line: median; whiskers: range. Trace scale bars: 0.1 ΔF/F; 0.5s.

Optogenetic CF co-activation differentially modulates basic responses to whisker stimulation in different types of neocortical neurons.

(A) Schematic of the recording configuration (left) and S1 cortex microcircuit (right). (B) CF co-activation with whisker stimulation significantly alters evoked responses of morphologically identified L2/3 pyramidal neurons. Left: averaged traces. Middle: analysis by neuron (AUC from 0-700 ms). Right: analysis by mouse. Blue line indicates mean. (C) CF co-activation significantly enhances responses in putative interneurons (INs; n=2014 cells; N=53 mice). (D – F) Sample two-photon field of view (left) and post-hoc verification of expression in S1 using confocal microscopy (right). Arrowheads indicate neurons co-expressing GCaMP6f and tdTomato in VIP interneurons (D), SST interneurons (E), or PV interneurons (F). Scale bars: 100µm (left); 500µm (right). (G) CF co-activation with whisker stimulation significantly reduces VIP population responses (n=129 cells, N=7 mice), but enhances responses in SST interneurons (n=102 cells; N=12 mice; H) and PV interneurons (n=177 cells; N=18 mice; I). Note VIP responses are reduced in a late analysis window (650-850 ms), while SST and PV responses are enhanced in the default 0-700 ms analysis window. Middle plots show analysis by neuron, right plots analysis by mouse (blue line indicates mean). Scale bars: 0.1 ΔF/F; 0.5s. Box: 25th and 75th percentiles; line: median; whiskers: range. Trace scale bars: 0.1 ΔF/F; 0.5s.

SST interneurons are recruited to prevent L2/3 pyramidal cell plasticity.

(A) Circuit diagram highlighting the chemogenetic activation of SST interneurons and recording configuration. (B) Expression of activating hM3D(Gq) receptors is localized to neocortical SST interneurons. Scale bar: 100µm (top) and 500µm (bottom). (C) DCZ application enhances responses in SST interneurons (n=12 cells; N=7 mice). (D) In the presence of DCZ, RWS stimulation suppresses L2/3 pyramidal cell potentiation, and even causes depression, even when the CF is not co-activated (n=255 cells; N=4 mice). Left: average traces; middle: AUC analysis by cell; right: maximum response amplitude by cell. (E) Circuit diagram highlighting the chemogenetic inhibition of SST interneurons and recording configuration. (F) Expression of inhibiting hM4D(Gi) receptors is localized to neocortical SST interneurons. Scale bars as in (B). (G) DCZ application reduces responses in SST interneurons (n=53 cells; N=6 mice). (H) In the presence of DCZ, pyramidal cell potentiation is observed, despite CF-co-activation (n=183 cells; N=3 mice). Data analysis presented as in (D). Box: 25th and 75th percentiles; line: median; whiskers: range. Trace scale bars: 0.2 ΔF/F; 0.5s.

VIP interneurons mediate the effects of optogenetic CF activation.

(A) Circuit diagram highlighting the chemogenetic inhibition of VIP interneurons and recording configuration. (B) Expression of inhibiting hM4D(Gi) receptors is localized to neocortical VIP interneurons. Scale bars: 100µm (top) and 500µm (bottom). (C) DCZ application reduces responses in VIP interneurons (n=10 cells; N=3 mice). (D) In the presence of DCZ, RWS stimulation suppresses L2/3 pyramidal cell potentiation, and even causes depression, even when the CF is not co-activated (n=189 cells; N=3 mice). Left: averaged traces; middle: AUC analysis by cell; right: maximum response amplitude by cell. (E) Circuit diagram highlighting the chemogenetic activation of VIP interneurons and recording configuration. (F) Expression of activating hM3D(Gq) receptors is localized to neocortical VIP interneurons. Scale bars as in (B). (G) DCZ application enhances responses in VIP interneurons (n=14 cells; N=3 mice). (H) In the presence of DCZ, pyramidal cell potentiation is observed, despite CF co-activation (n=202 cells; N=4 mice). Data analysis presented as in (D). Box: 25th and 75th percentiles; line: median; whiskers: range. Trace scale bars: 0.1 ΔF/F; 0.5s.

VIP interneurons orchestrate the inhibitory network in S1.

(A) Circuit diagram highlighting the chemogenetic inhibition of VIP interneurons and recording configuration. (B) Inhibiting hM4D(Gi) receptors are expressed in neocortical VIP interneurons. Note A-B are identical to Fig. 4.A-B. (C) In the presence of DCZ, RWS stimulation activates L2/3 interneurons, even when the CF is not co-activated (n=27 cells; N=3 mice). Left: averaged traces; middle: AUC analysis by cell; right: maximum response amplitude by cell. (D) Circuit diagram highlighting the chemogenetic activation of VIP interneurons and recording configuration. (E) Activating hM3D(Gq) receptors are expressed in neocortical VIP interneurons. Note D-E are identical to Fig. 4E-F. (F) In the presence of DCZ, IN depression is observed, despite CF co-activation (n=105 cells; N=4 mice). Data analysis presented as in (C). Box: 25th and 75th percentiles; line: median; whiskers: range. Trace scale bars: 0.2 ΔF/F; 0.5s.

A pathway from the cerebellar nuclei via the zona incerta and thalamic nucleus POm to S1 cortex.

(A) Schematic of dual-injection strategy to label outputs of zona incerta (ZI) neurons receiving input from contralateral cerebellar nuclei. (B) EYFP expression in the left hemisphere (left; scale bar: 1mm) with higher magnification images taken in the same slice. Center: EYFP expression in the ZI. Right: labeled axons in the posterior medial nucleus (POm; bottom; scale bar: 20µm) and S1 cortex (top; scale bar: 10µm). (C) Quantification of cerebellar nuclei projections to the ZI. Curves show terminal density distribution from dorsal ZI (dZI) to ventral ZI (vZI). Color of curves correspond to images taken along the rostro-caudal axis (green: top (rostral) image; navy: center image; light blue: bottom (caudal) image. Scale bars: 500µm. (D) Labeling strategy for electrophysiological recordings shown in (E-F). Scale bar: 1mm. (E) Whole-cell patch-clamp recordings from POm-projecting ZI neurons receiving input from cerebellar nuclei. Top: responses to depolarizing current pulses in the absence (left) and presence (right) of TTX (1µM). Bottom: Responses in ZI cells to photostimulation of ChR2-expressing terminals from cerebellar nuclei neurons in the absence (let) and presence (right) of TTX. (F) Amplitude (left) and latency (right) of photostimulation-evoked EPSPs before (n=5) and after (n=3) wash-in of TTX. (G) Experimental configuration for recordings from mice expressing inhibiting hM4D(Gi) receptors in PV-expressing ZI interneurons. (H) In the presence of DCZ, the suppressive effect of CF co-activation on acute whisker responses of L2/3 pyramidal neurons is blocked (n=80 cells; N=3 mice). (I) Bar graphs: analysis by neuron. Box: 25th and 75th percentiles; line: median; whiskers: range. Trace scale bars: 0.1 ΔF/F; 0.5s.

Optogenetic CF activation causes GCaMP6f-encoded calcium transients in Purkinje cells.

(A) Schematic of IO injection. Top left: Cerebellum (calbindin-staining of PCs) and IO (as in Fig 1B). Bottom: ChR2 expression in the principal IO (IOPr) and dorsal IO (IOD). Scale bars: 1mm (top) and 100µm (bottom). (B) Overlap of VGluT2-stained excitatory terminals with ChR2 expression in the contralateral cerebellar cortex. Scale bar: 40µm. (C) Post-hoc verification of ChR2 expression in CF terminals in crus I and II. Scale bars: 50µm. (D) Measure of distance from the pial surface to the cerebellar nuclei. While the IO innervates both the cerebellar cortex and nuclei directly, it is unlikely that CF terminals other than the superficial ones innervating PCs are optogenetically activated as the intensity of blue light decreases exponentially in brain tissue. Scale bar: 300µm. Dashed line: 2mm. (E) Experimental schematic for F-I. (F) Test of various stimulus protocols: 3×15ms, 2×20ms, 1×50ms (interval 50ms) and (3×15ms)*2 (interval 50ms). Traces show response averages (n=102 cells from one mouse). (G) Bar graphs showing response peaks (left) and integrals (right) from the corresponding data in F. Data: mean ± SEM. (H) Calcium transients evoked by 470nm, 50ms LED light pulses in PCs in crus I/II (n=131 cells; N=2 mice) in comparison to spontaneous events (left). Scale bars: 0.1 ΔF/F; 0.5s. (I) Amplitude and latency to peak measures from the corresponding data in H. Data: mean ± SEM. Scale bars: 0.1 ΔF/F; 0.5s.

Plasticity in different neuron types.

(A) Experimental schematic. (B) Event probability around stimulus onset with the window for detection of stimulus-evoked events (orange; three standard deviations above the mean event probability, calculated across all frames collected during the 20-second trial periods). (C) Pie chart indicating the percentage of L2/3 pyramidal cells recruited or suppressed by RWS/RWS+CF activity (only responsive to whisker stimulation in post- or pre- periods, respectively) and how many remained entirely unresponsive. (D) Left: RWS-evoked response changes in L2/3 pyramidal neurons (n=329 cells; N=12 mice). Right: RWS+CF-evoked response changes in L2/3 pyramidal neurons (n=597 cells; N=11 mice). Box plots quantify calcium responses across all cells and trials (i.e. including unresponsive neurons and trials without whisker-evoked responses). (E) The same as D in INs in the same mice (n=616 cells for RWS; n=590 cells for RWS+CF). (F, G) Same as (D, E) in tdTomato-tagged SST interneurons (F; n=41 cells and N=3 mice for RWS; n=31 cells and N=3 mice for RWS+CF) and PV interneurons (G; n=83 cells; N=5 mice for RWS; n=113 cells; N=5 mice for RWS+CF). Box: 25th and 75th percentiles; line: median; whiskers: range. Trace scale bars: 0.05 ΔF/F; 0.5s.

Impact of RWS- and RWS+CF-stimulation on animal motion.

(A) Experimental schematic. Mice are awake and head-fixed but can walk/run on the treadmill. (B) Average cross-correlation coefficient between neural calcium signals and movement activity data, segregated by neuron type. The numbers below the box charts indicate the number of recorded cells. (C) Left: probability distribution of animal movement in Rest trials, demonstrating the absence of movement both before and after stimulus onset. Right: probability distribution of animal movement in Active trials. (D) Pie charts indicating the percentage of Rest, Active and Stop trials pre-vs post-RWS (left) or RWS+CF (right). (E) Average probability of animal movement in Rest trials pre- and post-RWS (left) or RWS+CF (right) over the same time scale as the neural traces shown below. (F) and (H) RWS-evoked potentiation in L2/3 pyramidal neurons (n=124; N=12) and RWS+CF-mediated suppression of potentiation (n=388; N=11) in the absence of animal movement (i.e. in Rest trials). (G) and (I) Analysis for INs in the same mice (n=212 cells for RWS; n=296 cells for RWS+CF). Note motion activity traces capture both whisking and running activity. Box: 25th and 75th percentiles; line: median; whiskers: range. Trace scale bars: 0.2 ΔF/F; 0.5s.

Intrinsic optical imaging confirms CF-mediated suppression of S1 plasticity.

(A) Experimental schematic. Intrinsic optical imaging is performed using a high-speed camera. The field of view is illuminated with red light (625 nm). A decrease in ΔR/R indicates increased oxygen consumption by neurons activated by single whisker stimulation (blue arrow). (B) Top: Example of S1 plasticity evoked by stimulation of the C2 whisker at 10Hz. Bottom: Example of suppression of response potentiation when whisker stimulation is paired with optogenetic CF co-activation at 1Hz. (C) Quantification of RWS-evoked potentiation of the single whisker response area (blue; N=4 mice) and RWS+CF-mediated suppression of response potentiation (red; N=4 mice). No response potentiation is observed in the absence of repeated whisker stimulation or CF activation (grey; N=4 mice). Data are normalized to the pre-plasticity response area. Light blue line indicates mean.

Plasticity effects in L2/3 PNs vs controls in chemogenetic manipulation experiments.

(A) Chemogenetic activation of SSTs. Left: Experimental schematic. Middle: circuit diagram. Right: DCZ injection activates SSTs (yellow). Top: Control protocol. ‘Post’ marks the period equivalent in time to the period for the measurement of plasticity effects after RWS (bottom). (B) Responses in L2/3 PNs during Pre, DCZ wash-in, and Post periods in control experiments. (C) The difference in response (ΔAUC) of L2/3 pyramidal cells to whisker stimulation between the DCZ wash-in and Post periods; (control: n=218 cells; N=3 mice; plasticity: n=253 cells; N=4 mice); negative ΔAUC values indicate AUC values in the Post period are lower than in the DCZ wash-in period. The ΔAUC in control and plasticity experiments are normalized to the absolute ΔAUC in control experiments to demonstrate changes in the plasticity experiments relative to controls. Note the absence of PN potentiation in the plasticity experiment. (D) – (F) Equivalent measures during chemogenetic VIP inhibition (red; control: n=173 cells; N=3 mice; plasticity: n=164 cells; N=3 mice). Note the absence of PN potentiation in the plasticity experiment. (G) – (I) Equivalent measures during chemogenetic SST inhibition (control: n=175 cells; N= 3 mice; plasticity: n=181 cells; N=3 mice). (J) – (L) Equivalent measures during chemogenetic PV inhibition (control: n=368 cells; N=4 mice; plasticity: n=258 cells; N=3 mice). PNs are significantly potentiated after plasticity experiments beyond changes in controls. (m) – (o) Equivalent measures during chemogenetic VIP activation (control: 145 cells; N=3 mice; plasticity: n=197 cells; N=4 mice). PNs are significantly potentiated after plasticity experiments beyond changes in controls. Optogenetic CF co-activation is applied during RWS in the experiments shown in (G) – (O). Data: mean ± SEM.

Cerebellar nuclei do not strongly project to thalamic reticular nucleus.

(A) Schematic of labeling approach (top) and injection site of retrograde tracer (bottom). Scale bar: 1mm. (B) Image of retrograde label from the somatosensory thalamus (POm and VPM; cyan) and orthograde label from the contralateral cerebellar nuclei (red) taken approximately -1.0mm posterior from bregma, demonstrating lack of overlap between neurons in the thalamic reticular nucleus (TRN) and cerebellar nuclei terminals in the adjacent thalamic nuclei. Representative images are shown from two separate mice. Scale bars: 1mm (left images) and 250µm (right images). (C) Projection diagram. DCN: deep cerebellar nuclei; ZI: zona incerta; POm: posterior medial thalamic nucleus; TRN: thalamic reticular nucleus; VPM: ventral posterior medial nucleus; VPL: ventral posterior lateral nucleus; VA: ventral anterior nucleus; VL: ventral lateral nucleus.

Transsynaptic labeling identifies projections from the cerebellar nuclei to POm.

(A) Schematic of dual-injection approach to label outputs of ZI neurons receiving input from the contralateral CBN. (B) Top left: EYFP-expressing terminals and cell bodies within ZI (dashed lines) and axonal projections out of the ZI (arrowheads). Scale bar: 100µm. Bottom left: projections from the ZI terminate in POm (arrowheads). Scale bar: 50 µm. Right: zoomed-out view showing projections from ZI to POm in the same plane. Scale bar: 200 µm. (C) ZI neurons receiving cerebellar input project to S1. Synaptic terminals are observed in L1 and L5a. Scale bar: 100 µm. (D) Series of coronal sections showing details of the projections from the contralateral CBN to ZI and POm. Scale bars: 1mm. (E) The same as D in a second mouse, showing robust EYFP expression across the rostro-caudal extent of ZI. Scale bars: 1mm. POt = posterior thalamic nucleus (triangular part). Colored dots mark images taken from the same sections.

Whole-cell patch-clamp recordings are performed from neurons in the ZI that project to POm and receive input from cerebellar nuclei.

(A) Left: Differential interference contrast (DIC) image showing the ZI (white dashed circle) at low magnification (top; scale bar: 500µm), and a neuron selected for patching at higher magnification (bottom; scale bar: 100µm). Middle left: same images showing ChR2-expression. Middle right: same images showing retrograde fluoro-Ruby label originating in the POm. Right: merged images (boxed area with yellow dashed line marks the recordings area shown in the images below). (B) Post-hoc histology of an acute slice used for recordings demonstrating the details of cerebellar innervation of the ZI, separated by dorsal ZI (dZI) and ventral ZI (vZI; image corresponds to Fig. 6D, separated by channel). Scale bar: 500µm. (C) Quantification of the terminal fluorescence segregated by dZI and vZI. Data: mean ± SEM.

Data and statistical tests corresponding to Figure 1.

Data and statistical tests corresponding to Figure 2.

Data and statistical tests corresponding to Figures 3, 4, and 5.

Data and statistical tests corresponding to Figure 6.

Data and statistical tests corresponding to Supplementary Figure 1.

Data and statistical tests corresponding to Supplementary Figure 2.

Data and statistical tests corresponding to Supplementary Figure 3.

Data and statistical tests corresponding to Supplementary Figure 4.

Data and statistical tests corresponding to Supplementary Figure 5.

List of virus titers.