doi: 10.1038/s41467-020-19960-x.
A FN-MdV pathway and its role in cerebellar multimodular control of sensorimotor behavior
Affiliations
- PMID: 33247191
- PMCID: PMC7695696
- DOI: 10.1038/s41467-020-19960-x
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A FN-MdV pathway and its role in cerebellar multimodular control of sensorimotor behavior
Nat Commun.
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Abstract
The cerebellum is crucial for various associative sensorimotor behaviors. Delay eyeblink conditioning (DEC) depends on the simplex lobule-interposed nucleus (IN) pathway, yet it is unclear how other cerebellar modules cooperate during this task. Here, we demonstrate the contribution of the vermis-fastigial nucleus (FN) pathway in controlling DEC. We found that task-related modulations in vermal Purkinje cells and FN neurons predict conditioned responses (CRs). Coactivation of the FN and the IN allows for the generation of proper motor commands for CRs, but only FN output fine-tunes unconditioned responses. The vermis-FN pathway launches its signal via the contralateral ventral medullary reticular nucleus, which converges with the command from the simplex-IN pathway onto facial motor neurons. We propose that the IN pathway specifically drives CRs, whereas the FN pathway modulates the amplitudes of eyelid closure during DEC. Thus, associative sensorimotor task optimization requires synergistic modulation of different olivocerebellar modules each provide unique contributions.
Conflict of interest statement
The authors declare no competing interests.
Figures
a–c Schematic of the experimental design. a Head-fixed mouse is presented with a green LED light as the conditioned stimulus (CS) and a periorbital air puff as the unconditioned stimulus (US). b Conditioned responses (CRs, green) emerge over training days, prior to the onset of unconditioned responses (URs, red) during DEC training. c An example of DiI-labeled recording track in the cerebellum showing the electrophysiological recording location in the FN (n = 7 mice). Scale bar, 1 mm. d Activity of FN neurons during DEC. Top and middle rows: example traces of eyelid movement and single unit activity of FN neurons with CS-related facilitation (left) and suppression (right). Bottom row: group average of activity patterns for each modulation type (n = 86 for facilitation neurons; n = 16 for suppression neurons; mean ± s.e.m.). Green shading indicates the CS–US interval. e Fraction of FN neurons with different modulation types. f Heatmaps: an example cell with a positive correlation between neuron activity (left) and CR amplitude (right). Each row in the left heatmap represents a single trial recording and each row in the right heatmap represents the corresponding CR amplitude from the same trial. Trials are ordered from top to bottom by their peak facilitation amplitudes. Dashed lines indicate CS and US onsets. A positive trial-by-trial correlation between facilitation and CR amplitudes is shown in the right panel (linear regression model, P = 2.74e−7) and each dot represents a single trial. g Summary of all facilitation cells with a positive correlation (linear regression model, P < 0.05, n = 10). h Same as (f), but for a neuron with a negative trial-by-trial correlation between facilitation and CR amplitudes (linear regression model, P = 0.0003). i Same as (g), but for cells with a negative correlation (linear regression model, P < 0.05, n = 5). j Average correlation matrix of 86 facilitation FN cells. Each epoch indicates the mean r2 value of the trial-by-trial correlation between the FN neuron activity and eyelid closure at a given time point throughout the task. Most-correlated epochs (bright pixels) are located above the diagonal line and before US onset. CS and US onsets are denoted by dashed lines in both dimensions. k Summary of the relationship between facilitation onset and CR onset for all facilitation cells (mean ± SD, n = 86, paired two-sided t test, P = 1.05e−14). l Same as (k), but for the relationship of peak timings (mean ± SD, n = 86, paired two-sided t test, P = 0.0024).
a Schematics showing viral injection, optical fiber implantation and multichannel recording in the FN of the VGluT2-ires-Cre mice (n = 5) or the Gad2-ires-Cre mice (n = 8). b, Expression of Cre-dependent ChrimsonR in VGluT2-positive FN neurons (left), showing a short-latency response to 595 nm light (orange shading, right). The blue dashed line indicates the timing at which the firing rate exceeds three SDs of the baseline frequency within 20 ms after the light. Scale bar, 10 µm. c Same as (b), but for the Gad2-positive neurons. d Task-related modulation of VGluT2-positive neurons. Neurons are categorized based on their CS-related modulations. Top and middle rows: example eyelid movement and spike traces of individual cells. Bottom row: average firing rate of neurons with CS-related facilitation (left, n = 7), suppression (middle, n = 3) and no modulation (right, n = 5); traces are plotted as mean ± s.e.m. e Same as (d), but for the Gad2-positive neurons, showing no CS-related modulation (n = 8). f Left: experimental design for FN neuron recording and CTB tracing by using a single glass capillary. Right: a representative neuron showing CS- and US-related facilitation (overlaying eyelid closure, mean ± SD, n = 21 trials, upper right; PSTH, lower right) during the CS–US interval. g, h Iontophoresis of CTB localized to the recording site (g, left, scale bar, 1 mm) and retrogradely labeled Purkinje cells (g, right, scale bar, 20 µm) in the parasagittal vermis regions (h) (n = 5 mice).
a Representative DiI-labeled recording tracks in cerebellar vermal regions (lobules IV-VII). Scale bars, 1 mm. Experiments were performed with 17 mice. b Representative waveforms (mean ± coefficient of variation) of simple spikes and complex spikes from a single PC. c CS-related simple spike modulation in vermal PCs. Top and middle rows indicate example eyelid closure and spike traces of individual PCs (* indicates complex spikes); bottom: group average of simple spike activity from PCs of each modulation type (blue: PCs with simple spike suppression, n = 23; red: PCs with simple spike facilitation, n = 26/62), traces are plotted as mean ± s.e.m. d Fraction of PC population with simple spike modulations. e Example PC with a significant correlation between the simple spike suppression (left heatmap) and the CR peak amplitudes (right heatmap) over trials. Each row represents a single trial, ordered from bottom to top based on the magnitude of the simple spike suppression. The correlation of this cell is shown in the right panel (linear regression model, P = 1.24e−5), and each dot represents a single trial. f Summary of all PCs showing a significant trial-by-trial correlation between simple spike suppression and CR peak amplitude (linear regression model, P < 0.05, n = 8). g Average correlation matrix of 23 suppressed cells. Most-correlated epochs (bright pixels) are distributed across the diagonal line and before US delivery. CS and US onsets are denoted with dashed lines in both dimensions. h Comparison of the timing of simple spike suppression and behavior. Simple spike suppression precedes the CR both in onset (left, mean ± SD, n = 23, paired two-sided t test, ***P = 0.00013) and peak timing (right, mean ± SD, n = 23, paired two-sided t test, ***P = 1.94e−5).
a Complex spike modulation during DEC. PCs with CS-related complex spikes (CpxCS) are color-coded based on their simple spike (SS) modalities: suppression (CpxCS(SSS), blue), facilitation (CpxCS(SSF), red) and no modulation (CpxCS(SSN), gray). Top row: summary of eyelid responses (left to right: n = 30, 32, 41 trials, mean ± SD). Middle row: example complex spike activity (raster plots of spike events) during DEC, and bottom row shows average CpxCS activity of each PC population (left to right: n = 12, n = 12, n = 5 neurons, mean ± s.e.m.). b Comparison between the timing of CpxCS (CpxCS latency) and the CR onset. Only PCs with simple spike suppression showed an earlier occurrence of CpxCS than CR onset (mean ± SD, paired two-sided t test, left to right: n = 12, 12, and 5, P = 0.04, 0.51, and 0.14). c Comparison of CpxCS latency in trials divided into early (n = 16 trials, 147.4 ± 23.6 ms, mean ± SD) and late trials (n = 16 trials, 196.8 ± 21.2 ms, mean ± SD) based on CR onset. Example recording of CpxCS during the CS–US interval (firing rate PSTH, c, bottom) in the early and late CR trials (c, top). d Population summary showing no difference in CpxCS latency between early and late trials in any category of PCs (paired two-sided t test, P = 0.39). e Comparison of CR peak amplitudes in trials with and without CpxCS. The occurrence of CpxCS in the PCs with simple spike suppression predicts a larger CR amplitude (mean ± SD, paired two-sided t test, left to right: n = 12, 12, and 5, P = 0.005, 0.94, and 0.80). f Example traces of CRs (top, n = 21 trials for pink trace, n = 10 trials for green trace, mean ± SD) with or without CpxCS (firing rate PSTH, bottom). CpxCS is defined as the complex spikes that occur within 50–250 ms following CS onset. Correlation of CpxCS occurrence and CR peak amplitude for three categories of Purkinje cells is summarized in (g). PCs with simple spike suppression (CpxCS(SSs), blue) reside below the diagonal line.
a Example injection site of muscimol and alcian blue in the FN ipsilateral to the trained eye. Scale bar, 1 mm. b CR and UR performance of a mouse following muscimol inhibition of the FN. Average traces of eyelid movement, in control (black), by muscimol inhibition (cyan), and after washout (magenta) sessions from the same mouse. c Summary of the effects of muscimol inhibition on CR and UR performances (n = 3 mice, mean ± SD, paired two-sided t test, *P < 0.05, **P < 0.01). d Example cerebellar section showing exclusive expression of ChR2-tdTomato in the PCs of L7Cre-Ai27 mice. An optic fiber was implanted above the FN ipsilateral to the trained eye. Scale bar, 1 mm. e, f Same as (b, c), but for the optogenetic perturbation of FN neurons during the CS–US interval (indicated in blue bar). Both CRs and URs were suppressed (n = 5 mice, mean ± SD, paired two-sided t test, *P < 0.05, **P < 0.01). g–i Same as (d–f), but for optogenetic perturbation of the simplex lobule-IN module. CRs, but not URs, were suppressed (n = 3 mice, mean ± SD, paired two-sided t test, *P < 0.05, **P < 0.01). j Example image of cerebellar section after laser photolesion. Dashed contour highlights the strong autofluorescence from FN lesion site (n = 4 mice). Scale bar, 1 mm. k Representative CR traces from a trained mouse before (black) and after FN lesion (red). l Summary of CR-trial probabilities in the control (n = 5, mean ± s.e.m., black trace) and FN lesion groups (n = 4, mean ± s.e.m., red trace). CR-trial probability in lesion animals was lower than that of the control group (two-way repeated measures ANOVA, *P < 0.05). Dashed line indicates the time point for the photolesion. m Same as (l), but for the comparison of CR amplitudes in two groups. n Comparison of the CR-trial probability between the pre-lesion session (day 11) and 3 post-lesion sessions (days 12–14), in lesion group (red, n = 4, two-way repeated measures ANOVA, *P < 0.05) and control group (black, n = 5, paired two-sided t test, P > 0.05). Dots and lines indicate performance of different mice, mean ± s.e.m. o Same as (n), but for the comparison of CR amplitudes before and after lesion in the lesion group (n = 4, paired two-sided t test, *P < 0.05, **P < 0.01) and control group (n = 5, P > 0.05). See the exact P values for each comparison in the Source Data file.
a Experimental design for chemogenetic inhibition of the FN during DEC training (left). Inhibitory DREADD-hM4D (Gi) was expressed in FN ipsilateral to the trained eye (right, n = 6 mice) and tdTomato was expressed in control mice (n = 8). Both control and DREADD mice received a i.p. CNO injection 15–20 min prior to training. Scale bar for the inserted image is 50 µm. b Representative FN neuron responses from control and DREADD-expressing animals at early (left) and late (right) stages of recording, following the CNO injection. c Comparison of neuron activity over time after the CNO injection in control (n = 37 neurons) and DREADD-expressing (n = 19 neurons) mice (two-way repeated measures ANOVA, ***P < 0.001). d Progression of CR traces during DEC training in a representative DREADD mouse (CS–US interval shown in red) and a control mouse (CS–US interval shown in cyan). e Comparison of the CR acquisition during training (1–10 days), illustrated in the CR peak amplitude (upper) and CR-trial probability (lower), in control (n = 8 mice, mean ± s.e.m.) and DREADD-expressing mice (n = 6 mice, mean ± s.e.m., maximum likelihood estimation (two-sided), **P < 0.01, ***P < 0.001). f Comparison of CR performance on the 11th day with the CNO injection omitted (mean ± s.e.m., two-sample t test (two-sided), *P < 0.05, **P < 0.01). g–i Same as in (d–f), but for DEC training in mice with optogenetic perturbation. CR acquisition is suppressed in L7Cre-Ai27 mice (n = 4 mice, mean ± s.e.m.) compared to the acquisition observed in the control group (n = 5 mice, mean ± s.e.m., maximum likelihood estimation (two-sided), ***P < 0.001). Comparison of CR performance on the 11th day with opto-inhibition omitted (mean ± s.e.m., two-sample t test (two-sided), **P < 0.01). See the exact P values for each comparison in the Source Data file.
a Experimental design of 7N neuron recording during DEC from L7cre-Ai27 mice with either IN (left) or FN (right) inhibition. Arrow-headed lines indicate outputs from the IN and the FN to 7N motor neurons. b Putative 7N motor neuron activity during DEC and spontaneous eyelid movements. Left: example recording of eyelid movement (upper) and a 7N neuron (lower) showing spike rate increases in response to the DEC triggers (marked with CS and US) as well as spontaneous eyelid movements (red arrowheads). Right: average relative firing rate of all putative 7N motor neurons (n = 19, mean ± s.e.m) during spontaneous blinking (peak-aligned, red arrowhead). c–e Changes in behavior and 7N neuron activity in the control, IN-inhibition and FN-inhibition trials. Firing rate PSTH of an example 7N neuron (middle row) during behavior (upper row, n = 46, 22, 26 trials for c–e), showing that both IN and FN photoperturbations (blue bars) inhibited CR and neuron activity in response to CS, while only FN-inhibition suppressed UR and neuron activity in response to US. Lower: average spike modulation of all 7N neurons (n = 19, mean ± s.e.m). Dashed-line trace indicates the average activity during control trials in (c). f Summary of changes in relative firing rates of 7N motor neurons in response to CS and US in control, IN-opto and FN-opto trials (n = 19, mean ± s.e.m., paired two-sided t test, **P < 0.01, ***P < 0.001). CR and UR modulation (∆Firing rate) are normalized to the corresponding firing rate changes of the control session. See the exact P values for each comparison in the Source Data file.
a Sketch of the viral tracing strategy (left). Middle and right images: coronal sections of the injection sites from an example animal. Retrograde GFP and anterograde RFP were simultaneously injected into the ipsilateral facial nucleus (7N, middle) and FN (right). Labeled fibers in the genu of facial nucleus (g7) confirm the targeting of the 7N. Scale bars, 1 mm. b Representative images of retrogradely labeled neurons (green) and anterogradely labeled FN axons (red) at the level of caudal midbrain. The raw image (left) is registered to the Allen Mouse Brain CCF (middle, see “Methods”) for further quantification of the FN projection in the contralateral red nucleus (right image, the RN is denoted by a dashed-line contour). Scale bars, 500 µm, inserted image 50 µm. c Same as (b), but for labeling in caudal medullary regions. Colocalizations of 7N projecting neurons and FN axons are found in the contralateral ventral medullary reticular nucleus (MdV, arrowheads in the right image). Scale bars, 500 µm, inserted image 50 µm. d Comparison of colocalizations in the RN and MdV from 4 mice (mean ± s.e.m., paired two-sided t test, *P = 0.020). e Confocal images of two example MdV neurons with FN axons targeting their primary dendrite (top) and soma (bottom). Scale bars, left column, 20 µm, right columns,10 µm. Tracing experiments were performed and replicated in n = 4 mice.
a Schematics showing viral injections and the optogenetic inhibition strategy to selectively identify MdV-projecting FN neurons and suppress the FN-MdV pathway during DEC. b Example image showing the somatic-targeting inhibitory opsin, stGtACR2, exclusively in FN neurons (insert). Scale bars, 200 µm, inserted image 10 µm, n = 6 mice. c FN neuron activity during optogenetic stimulation. Top: raster plot of an example neuron showing spike inhibition in response to optic light (n = 13 trials); bottom: average firing rate of all FN neurons in response to optic light (n = 15 neurons, mean ± s.e.m.). d Activity of the same photo-identified MdV-projecting FN neurons (n = 15 neurons, mean ± s.e.m.) in (c) during DEC. e, f Effects of optogenetic suppression of the FN-MdV pathway on behavior. Left column: average behavioral traces of an example mouse during CS-only trials (e) and CS–US paired trials (f). Middle and right columns: summary of behavioral performance. CR and UR amplitudes are significantly suppressed in trials with optogenetic perturbation (n = 6 mice, mean ± SD, paired two-sided t test, **P < 0.01), while UR timing is not influenced compared to control trials (n = 6 mice, mean ± SD, paired two-sided t test, P > 0.05). g Schematic drawing showing the proposed organization of the interposed nucleus (IN) and the fastigial nucleus (FN) pathways for DEC. Solid lines denote direct projections, and dashed lines indicate indirect innervations. Abbreviations: 7N, facial nuclei; CF, climbing fiber; GC, granule cell; IO, inferior olive; MdV, ventral medullary reticular nucleus; MF, mossy fiber; PN, pontine nuclei; RN, red nuclei. See the exact P values for each comparison in the Source Data file.
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