GlyT2-Positive Interneurons Regulate Timing and Variability of Information Transfer in a Cerebellar-Behavioral Loop

Abstract

GlyT2-positive interneurons, Golgi and Lugaro cells, reside in the input layer of the cerebellar cortex in a key position to influence information processing. Here, we examine the contribution of GlyT2-positive interneurons to network dynamics in Crus 1 of mouse lateral cerebellar cortex during free whisking. We recorded neuronal population activity using Neuropixels probes before and after chemogenetic downregulation of GlyT2-positive interneurons in male and female mice. Under resting conditions, cerebellar population activity reliably encoded whisker movements. Reductions in the activity of GlyT2-positive cells produced mild increases in neural activity which did not significantly impair these sensorimotor representations. However, reduced Golgi and Lugaro cell inhibition did increase the temporal alignment of local population network activity at the initiation of movement. These network alterations had variable impacts on behavior, producing both increases and decreases in whisking velocity. Our results suggest that inhibition mediated by GlyT2-positive interneurons primarily governs the temporal patterning of population activity, which in turn is required to support downstream cerebellar dynamics and behavioral coordination.

Keywords: cerebellum; chemogenetics; computation; inhibition; interneuron; neural populations.

Figure 1.

Single unit and population activity in Crus 1 during voluntary whisking. a, Monitoring whisker movement and neuronal population activity in lateral cerebellar cortex using high-speed videography and Neuropixels recording in head-fixed mice. Left, Whiskers were labeled and reconstructed using DeepLabCut to recover movement trajectories. Image credit: Elisabeth Meyer. Right, Bright-field image of coronal cerebellar section superimposed fluorescent image tract left by Neuropixels probe coated with DiI localized to lobule Crus 1. b, 30 s segment of simultaneous whisking (top) and population activity (bottom; n = 88 units) from a single recording session. c, Trial-averaged whisker position and neuronal activity from the same recording, aligned to onset of whisking bout. d, Tuning curves of four individual cerebellar units from the same recording. Each unit displays a different relationship between firing rate and whisker position. Blue lines indicate fraction of time spent at different whisker positions; empty gray bars show tuning curves for shuffled data. Left inset, Color-coded center of mass of each unit on Neuropixels probe. e, Average tuning curve for all recorded units, from all recordings (black line; n = 508, N = 12). Population firing rate increases monotonically from whisker resting point (white triangle) in both protractive and retractive directions. Blue line indicates fraction of time spent at different whisker positions; shading represents standard deviation; white triangle indicates whisker resting point; gray line shows tuning for shuffled data. f, Average tuning curves for all units clustered in eight groups. Units were clustered using k-mean algorithm based on individual unit tuning curves. Heterogeneity in tuning curves enables a continuous encoding of whisking position. g, Single trial of whisker movement (top) with corresponding projections in 3D principal component space for average (bottom left) and single trial (bottom right) population activity. Population activity in this space has structure that reflects whisking dynamics, both on average and at a single trial level. h, Recovered movement trajectory using cerebellar population activity. Reconstruction of the whisking set point using a linear combination of the first three principal components computed from neuronal populations. Whisker set point (brown line) during two trials, together with predictions (red) and the highest density interval (hdi, red shading).

Figure 2.

Population representations of whisker movement at different timescales. a, Trial-averaged whisking position (top) and projected population activity (bottom) for one recording. b, Cross-correlations between pc1–3 and whisking activity for the same recording; vertical lines indicate times of peak correlation. c, Cumulative variance explained by pc1–3 across all recordings (N = 12). Only a modest amount of variability in neuronal activity is accounted for by first three principal components. d, Distribution of unit loadings (n = 508) for pc1–3. Kurtosis measurement, to quantify the number of outliers in each distribution, reveals fewer outliers for pc1 than what would be expected if the data were normally distributed (excess kurtosis −2.76). This indicates that information contained in pc1, which best reflects whisking activity, tends to be distributed across neurons. The loading distributions for pc2 and pc3 have respectively a similar or higher number of outliers compared with normally distributed data (excess kurtosis −0.43 and 4.97, respectively), indicating that information in pc2 and pc3 is increasingly concentrated in fewer units. e, Time of peak correlation for pc1–3 across all recordings. The distribution for pc1 is tightly concentrated around ∼40 ms, meaning that information captured by pc1 tends to anticipate whisking activity with high temporal precision (one-sample t test; t = 4.71; p = 0.0006). The information contained in pc2 and pc3 shows the opposite trend, with more variability (pc2: mean −194 ms, t = −4.44, p = 0.0009; pc3: mean −92 ms, t = −1.44, p = 0.17), suggesting that these components might reflect different aspects of behavior. f, Mean decrease in unexplained variance (unexp. var.) across recordings with increasing numbers of principal components (#pcs); standard deviation shaded in gray.

Figure 3.

Chemogenetic inhibition of GlyT2-positive cells increases neuronal activity in cerebellar cortex. a, Left, Targeted expression of hM4Di receptors was achieved via injection of AAV-DIO-hM4Di-mCherry into the lateral cerebellar cortex of GlyT2-Cre mice, selectively expressing Cre-recombinase in GlyT2-positive cells in this brain region. Right, Neuropixels probes were targeted to site of viral injection. During electrophysiological recordings, the exogenous drug, clozapine N-oxide (CNO) was topically applied to activate hM4Di receptors. b, Left, GlyT2-Cre mouse brain selectively expressing hM4Di in GlyT2-positive cells of the lateral cerebellar cortex. Right, Tract left by Neuropixels probe coated with DiI, showing colocalization of the site of recording and hM4Di expression. c, Changes in population spike count before and after CNO/vehicle delivery (at 0 min) normalized by count at −5 min, for three experiment conditions. Pink, GlyT2-Cre mice and CNO (N = 19); blue, C57BL6 mice and CNO (N = 5); green, GlyT2-Cre/C57BL6 mice and saline vehicle only (N = 9). d, Box plots showing pooled data for each experimental condition. e, Multilevel modeling approach to assess effect of drug delivery on cerebellar population activity. Population spike count distributions for each condition (top) were modelled using an inverse-Gamma distribution, described by shape parameter α and scale parameter β. Each parameter was modeled as a linear combination of different coefficients, including two θ_cond, one for α and one for β, which captured the specific effect of the experimental manipulation on total spike counts. Comparing the empirical distribution with the distribution of posterior predictive checks (samples from the model after fitting) shows that the model captures the overall structure of the data (bottom). f, 94% highest density intervals (hdi) of the contrasts between post- and predrop posterior samples for θ_cond for α and β. The contrasts highlight a specific effect of GlyT2-positive cell manipulation (GlyT2 + CNO) on both α and β parameters. g, Contrast difference (mean and variance) between post- and predrop of the inverse-Gamma distribution fitted to total spike counts. Bars indicate the 94% hdi of the posterior differences. Left, Contrast difference between GlyT2 + CNO condition and each control condition. Right, Contrast difference between the two control conditions. GlyT2-positive cell manipulation produces positive contrast differences versus both control conditions.

Figure 4.

Weak influence of GlyT2-positive cell perturbation on cerebellar sensorimotor representations. a, Average tuning curves for GlyT2-CNO recordings before (gray) and after (red) topical administration of the drug. b, Entropy cumulative density functions for all units recorded in the GlyT2-CNO (top) and control (bottom) conditions. Under control conditions, but not GlyT2-CNO, unit entropy tends to increase after drug/vehicle delivery (postdrop), suggesting that tuning curves become less informative about whisking position over time. c, Probability mass function of unit entropy differences before and after CNO/vehicle administration (“postdrop” minus “predrop” entropy). Under control conditions, tuning curve entropy tends to remain stable or increase over time (density skewness, 2.79). In the GlyT2-CNO recordings, a fraction units exhibit decreased entropy after drug application (density skewness, 0.21), suggesting that some units become more sensitive to changes in whisking position following GlyT2-positive cell inhibition. d, Comparison of peak pre- and postdrop correlation values between principal components 1–3 (pc1–3) and whisker position for control recordings. e, Comparison of peak pre- and postdrop correlation values between principal components 1–3 (pc1–3) and whisker position for GlyT2-CNO recordings.

Figure 5.

GlyT2-positive cell inhibition reduces temporal variability in neuronal populations and weakens behavioral coupling at movement onset. a, Peri-event time histograms aligned to onset of whisker movement for pre- (top) and postdrop (bottom) periods for a GlyT2-CNO recording. Gray and red dots indicate the absolute peak of neuronal activity centered around whisking onset for the pre- and postdrop period, respectively. Units ranked according to their peak firing rate. Temporal jitter peak activity decreases after CNO application. b, Temporal dispersion of neuronal population activity before and after drug/vehicle administration (pre- and postdrop) for control (n = 12) and GlyT2-CNO (n = 13) recordings. Each data point represents the standard deviation of the distribution of absolute peak times of neuronal activity for each recorded population. Reduction of GlyT2-positive cell inhibition decreases the temporal dispersion of neuronal activity during whisking initiation (two-sided Wilcoxon signed-rank test; T = 12; p = 0.017). c, Average whisking onsets for two representative GlyT2-CNO recordings before (gray) and after (red) drug administration. Dashed lines represent linear fits of initial whisker protraction. CNO delivery was associated with both increased (top) and decreased (bottom) slope of protraction. d, Change in slope of whisker protraction at movement onset before and after drug/vehicle administration (pre- and postdrop) for control and GlyT2-CNO recordings. e, Box plot showing differences in slope of whisker protraction at movement onset between pre- and postdrug/vehicle administration for control and GlyT2-CNO recordings. The standard deviation of the slope distribution in the GlyT2-CNO condition is higher than controls (Levene's test W = 8.39; p = 0.008) suggesting that decreasing local GlyT2-positive cell inhibition produces variable changes in the dynamics of movement onset across recordings. f, Relationship between change in cerebellar population dynamics and whisker movement following drug/vehicle administration for all recordings. Under control conditions, measured changes in neural activity and movement are small in magnitude and well correlated (r² = 0.52). In GlyT2-CNO recordings, relationships between cerebellar activity and movement are decoupled (r² = 0.15).